Mutant rb69 dna polymerase

ABSTRACT

Provided herein are mutant DNA-dependent polymerases which are derived from, or otherwise related to, wild type RB69 DNA polymerase. These mutant polymerases are capable of selectively binding labeled nucleotides. These mutant polymerases are also capable of incorporating a variety of naturally occurring and modified nucleotides, including, for example, terminator nucleotides.

This application is a divisional of U.S. Nonprovisional application Ser.No. 14/191,997, filed on Feb. 27, 2014, which is a continuation of U.S.Nonprovisional application Ser. No. 13/600,416, filed on Aug. 31, 2012,now issued U.S. Pat. No. 8,703,461, which is a continuation of U.S.Nonprovisional application Ser. No. 12/790,768, filed on May 28, 2010,now abandoned, which claims the filing date benefit of U.S. ProvisionalApplication No. 61/263,320, filed on Nov. 20, 2009; and the subjectapplication is a continuation of U.S. Non-provisional application Ser.No. 14/991,230, filed Jan. 8, 2016, which is a continuation of U.S.Non-provisional application Ser. No. 14/108,166, filed on Dec. 16, 2013,now issued U.S. Pat. No. 9,255,258, which is a continuation of U.S.Non-provisional application Ser. No. 12/790,760, filed on May 28, 2010,now issued U.S. Pat. No. 8,632,975, which claims the filing date benefitof U.S. Provisional Application No. 61/184,774, filed on Jun. 5, 2009,and 61/242,762, filed on Sep. 15, 2009, and 61/295,533, filed on Jan.15, 2010. The contents of each of the foregoing patent applications areincorporated by reference in their entirety for all their purposes.

FIELD

The disclosure relates generally to mutant DNA-dependent polymeraseswhich are derived from or otherwise related to RB69 polymerases. Themutant RB69 polymerases disclosed herein are capable of binding and/orincorporating labeled nucleotides. Typically, the nucleotide bindingand/or incorporation properties of these mutant polymerases are altered,e.g., increased or decreased, relative to the corresponding nucleotidebinding and/or incorporation properties of wild type RB69 polymerase.

BACKGROUND

DNA polymerases typically catalyze nucleic acid synthesis usingpolynucleotide templates and employ Watson-Crick base pairinginteractions between the template-based-nucleotide and incomingnucleotides which bind at the polymerase active site. DNA polymerasesare useful in a variety of biological applications, including DNAsequencing applications.

Certain single molecule DNA sequencing methods encompass two steps,relying on nucleotide transient-binding to the polymerase, instead ofnucleotide incorporation (e.g., Vander Horn, et al., U.S. Ser. No.61/184,774; 61/242,763; and 61/295,533). The first step includestransiently-binding an incoming nucleotide (e.g., labeled nucleotide)with a polymerase under conditions which inhibit incorporation of thebound nucleotide, and the identifying the transiently-bound nucleotide.The second step includes incorporating a single nucleotide (e.g.,terminator nucleotide) so as to translocate the polymerase to the nextposition on the DNA template. The transiently-bound nucleotide can bereplaced with the nucleotide to be incorporated.

It is desirable to perform the first and second steps under moderatetemperature conditions (e.g., room temperature) with one type of DNApolymerase. It is also desirable to use a DNA polymerase which bindsincorporating incoming nucleotides that are complementary to thetemplate-based-nucleotide. It is also desirable to use a DNA polymerasewhich exhibits increased transient-binding duration (without nucleotideincorporation) for the incoming nucleotides, to increase the bindingduration of a labeled nucleotide to a polymerase, so as to increasedetection and identity of the bound nucleotide.

However, existing polymerases cannot be used to perform the first andsecond steps because they lack certain properties. For example, many DNApolymerases do not selectively bind labeled nucleotides. Similarly, manyDNA polymerases do not efficiently incorporate terminator nucleotides.Some DNA polymerases can exhibit short nucleotide binding duration, orcan catalyze nucleotide incorporation under conditions when transientnucleotide binding is desired. Additionally, some DNA polymerases canexhibit undesirable behaviors such as binding non-complementary incomingnucleotides, or incorporating non-complementary incoming nucleotides.

Thus, existing polymerases offer limited utility for conducting certaintwo-step, single molecule sequencing methods. These and other desirableproperties can be enhanced via modifying and selecting a DNA polymerase.Provided herein are mutant RB69 DNA polymerases which are useful forconducting these two-step DNA sequencing methods. In some embodiments,these mutant DNA polymerases can selectively and transiently bindlabeled nucleotides. In some embodiments, the mutant DNA polymerases canincorporate terminator nucleotides.

SUMMARY

Provided herein are novel DNA polymerase compositions, methods of makingsuch compositions and methods of using such compositions in variousbiological applications.

Provided herein are isolated mutant DNA polymerases capable ofselectively transiently-binding a labeled nucleotide and selectivelyincorporating a terminator nucleotide. In one embodiment, the isolatedmutant DNA polymerase comprises a mutant RB69 DNA polymerase. In anotherembodiment, the isolated mutant DNA polymerase comprises the amino acidsequence according to any one of SEQ ID NOS:1-8. In another embodiment,the isolated mutant DNA polymerase further comprises a reporter moiety.In another embodiment, the reporter moiety can be an energy transferdonor moiety. In another embodiment, the energy transfer donor moietycan be a fluorescent dye or a nanoparticle.

Also provided herein are systems comprising a mutant DNA polymerasebound to a DNA template and a primer, and a nucleotide transiently-boundto the mutant DNA polymerase, where the mutant DNA polymerase may becapable of selectively transiently-binding a labeled nucleotide andselectively incorporating a terminator nucleotide. In one embodiment,the system comprises a mutant DNA polymerase which can be a mutant RB69DNA polymerase. In another embodiment, the system comprises a mutant DNApolymerase having the amino acid sequence according to any one of SEQ IDNOS:1-8. In another embodiment, the system comprises a mutant DNApolymerase which further includes a reporter moiety. In anotherembodiment, the reporter moiety can be an energy transfer donor moiety.In another embodiment, the energy transfer donor moiety can be afluorescent dye or a nanoparticle.

Also provided are nucleic acid molecules encoding the mutant DNApolymerase which may be capable of selectively transiently-binding alabeled nucleotide and selectively incorporating a terminatornucleotide. In one embodiment, the nucleic acid molecule can be DNA orRNA.

Also provided herein are vectors, comprising the nucleic acid moleculewhich encodes the mutant DNA polymerase which may be capable ofselectively transiently-binding a labeled nucleotide and selectivelyincorporating a terminator nucleotide. In one embodiment, the vectorfurther comprising a promoter sequence joined with the nucleic acidmolecule encoding the mutant DNA polymerase. In one embodiment, thepromoter can be constitutive or inducible.

Also provided herein are host cells carrying the vector which comprisesthe nucleic acid molecule which encodes the mutant DNA polymerase whichmay be capable of selectively transiently-binding a labeled nucleotideand selectively incorporating a terminator nucleotide. In oneembodiment, the host cell can be a phage, a prokaryote cell or aeukaryote cell.

Also provided herein are methods for producing a mutant DNA polymerasepolypeptide, comprising culturing host cells under conditions suitablefor the host cell to produce the mutant DNA polymerase polypeptide. Inone embodiment, the host cells carry the vector which comprises thenucleic acid molecule which encodes the mutant DNA polymerase which maybe capable of selectively transiently-binding a labeled nucleotide andselectively incorporating a terminator nucleotide. Also provided aremutant DNA polymerase produced by this method.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-C show the full-length nucleotide and amino acid sequences ofnative RB69 polymerase (amino acid: SEQ ID NO:1) (nucleotide: SEQ IDNO:9).

FIGS. 2A-C show the full-length nucleotide and amino acid sequences ofan exemplary mutant RB69 polymerase called “3PDX” according to thepresent disclosure (amino acid: SEQ ID NO:2) (nucleotide: SEQ ID NO:10).

FIGS. 3A-C show the full-length amino acid sequences of an exemplarymutant RB69 polymerase called “SX” according to the present disclosure(amino acid: SEQ ID NO:3).

FIGS. 4A-C show the full-length amino acid sequences of an exemplarymutant RB69 polymerase called “DX” according to the present disclosure(amino acid: SEQ ID NO:4).

FIGS. 5A-C show the full-length amino acid sequences of an exemplarymutant RB69 polymerase called “FDX” according to the present disclosure(amino acid: SEQ ID NO:5).

FIGS. 6A-C show the full-length amino acid sequences of an exemplarymutant RB69 polymerase called “PDX” according to the present disclosure(amino acid: SEQ ID NO:6).

FIGS. 7A-C show the full-length amino acid sequences of an exemplarymutant RB69 polymerase called “FPDX” according to the present disclosure(amino acid: SEQ ID NO:7).

FIGS. 8A-C show the full-length amino acid sequences of an exemplarymutant RB69 polymerase called “F3PDX” according to the presentdisclosure (amino acid: SEQ ID NO:8).

FIG. 9A shows a stopped-flow fluorescence trace (t_(pol)) for Phi29(exo−) polymerase and terminal phosphate labeled dN4P nucleotides in thepresence of manganese (Example 2).

FIG. 9B shows a stopped-flow fluorescence trace (t_(pol)) for Phi29(exo−) polymerase and terminal phosphate labeled dN4P nucleotides in thepresence of calcium (Example 2).

FIG. 9C shows a stopped-flow fluorescence trace (t⁻¹) for Phi29 (exo−)polymerase and terminal phosphate labeled dN4P nucleotides in thepresence of manganese (Example 2).

FIG. 9D shows a stopped-flow fluorescence trace (t⁻¹) for Phi29 (exo−)polymerase and terminal phosphate labeled dN4P nucleotides in thepresence of calcium (Example 2).

FIG. 9E shows stopped-flow fluorescence traces for Phi29 (exo−)polymerase and terminal phosphate labeled dN4P nucleotides in thepresence of calcium (binding with dG4P) or manganese (chasing withEDTA+NaCl) (Example 2).

FIG. 9F shows stopped-flow fluorescence traces (t_(pol)) for Phi29(exo−) polymerase, and correct and incorrect terminal phosphate labeleddN4P nucleotides, in the presence of calcium (Example 2).

FIG. 9G shows stopped-flow fluorescence titration traces (t_(pol)) forPhi29 (exo−) polymerase and increasing amounts of the correct terminalphosphate labeled dN4P nucleotide in the presence of calcium (Example2).

FIG. 9H shows data from FIG. 7E which is fitted to a hyperbola equationto extrapolate the apparent nucleotide dissociation constant (k_(d)^(app)) for Phi29 DNA polymerase (see Example 2).

FIG. 10A shows a stopped-flow fluorescence trace (t_(pol)) for RB69(exo−) polymerase and terminal phosphate labeled dN4P nucleotides in thepresence of manganese (Example 2).

FIG. 10B shows a stopped-flow fluorescence trace (t_(pol)) for RB69(exo−) polymerase and terminal phosphate labeled dN4P nucleotides in thepresence of calcium (Example 2).

FIG. 10C shows a stopped-flow fluorescence trace (t⁻¹) for RB69 (exo−)polymerase and terminal phosphate labeled dN4P nucleotides in thepresence of manganese (Example 2).

FIG. 10D shows a stopped-flow fluorescence trace (t⁻¹) for RB69 (exo−)polymerase and terminal phosphate labeled dN4P nucleotides in thepresence of calcium (Example 2).

FIG. 10E shows stopped-flow fluorescence traces (t_(pol)) for RB69(exo−) polymerase, and correct and incorrect terminal phosphate labeleddN4P nucleotides, in the presence of calcium (Example 2).

DETAILED DESCRIPTION

Provided herein are mutant DNA-dependent polymerases which are derivedfrom, or otherwise related to, wild type RB69 DNA polymerase. Thesemutant polymerases are capable of selectively binding labelednucleotides. These mutant polymerases are also capable of incorporatinga variety of naturally occurring and modified nucleotides, including,for example, terminator nucleotides, dideoxynucleotides,acyclo-nucleotides, 3′ modified nucleotides (e.g., 3′ azido-modifiednucleotides), and/or ribonucleotides. These mutant RB69 polymerases canbe used for various nucleotide incorporation methods and sequencingmethods, including but not limited to the methods disclosed in U.S. Ser.No. 61/164,324, filed on Mar. 27, 2009; 61/184,774, filed on Jun. 5,2009; and 61/242,762, filed on Sep. 15, 2009.

Also provided herein are DNA sequencing methods using the mutant DNApolymerases. The methods encompass two steps: a nucleotide binding step,and a nucleotide incorporation step. In the nucleotide binding step, thenucleotide binds the active site of the polymerase and dissociates as anintact nucleotide (i.e., no cleavage and release of the phosphategroups).

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of ordinary skillin the art to which these inventions belong. All patents, patentapplications, published applications, treatises and other publicationsreferred to herein, both supra and infra, are incorporated by referencein their entirety. If a definition and/or description is explicitly orimplicitly set forth herein that is contrary to or otherwiseinconsistent with any definition set forth in the patents, patentapplications, published applications, and other publications that areherein incorporated by reference, the definition and/or description setforth herein prevails over the definition that is incorporated byreference.

The practice of the present disclosure will employ, unless otherwiseindicated, conventional techniques of molecular biology, microbiologyand recombinant DNA techniques, which are within the skill of the art.Such techniques are explained fully in the literature. See, for example,Sambrook, J., and Russell, D. W., 2001, Molecular Cloning: A LaboratoryManual, Third Edition; Ausubel, F. M., et al., eds., 2002, ShortProtocols In Molecular Biology, Fifth Edition.

As used herein, the terms “comprising” (and any form or variant ofcomprising, such as “comprise” and “comprises”), “having” (and any formor variant of having, such as “have” and “has”), “including” (and anyform or variant of including, such as “includes” and “include”), or“containing” (and any form or variant of containing, such as “contains”and “contain”), are inclusive or open-ended and do not excludeadditional, unrecited additives, components, integers, elements ormethod steps.

As used herein, the terms “a,” “an,” and “the” and similar referentsused herein are to be construed to cover both the singular and theplural unless their usage in context indicates otherwise. Accordingly,the use of the word “a” or “an” when used in the claims orspecification, including when used in conjunction with the term“comprising”, may mean “one,” but it is also consistent with the meaningof “one or more,” “at least one,” and “one or more than one.”

As used herein, the term “operably linked” and its variants refer tochemical fusion or bonding or association of sufficient stability towithstand conditions encountered in the nucleotide incorporation methodsutilized, between a combination of different compounds, molecules orother entities such as, but not limited to: between a mutant polymeraseand a reporter moiety (e.g., fluorescent dye or nanoparticle), orbetween a nucleotide and a reporter moiety (e.g., fluorescent dye).

As provided herein, the terms “polymerase” and “polymerases” arebiologically active polypeptide molecules, or fragments thereof, thatcatalyze transfer of a nucleoside monophosphate from a nucleosidepolyphosphate (or analog thereof) to the terminal 3′ hydroxyl group ofthe polymerization initiation site (i.e., nucleotide polymerization).The terminal 3′ hydroxyl group of a primer, or a gap or nick, or of aself-priming template, provides the polymerization initiation site forDNA polymerase.

Other objects, features and advantages of the disclosed methods, systemsand compositions will become apparent from the following detaileddescription. It should be understood, however, that the detaileddescription and the specific examples, while indicating specificembodiments, are given by way of illustration only, since variouschanges and modifications within the spirit and scope of the inventionsprovided herein will become apparent to those skilled in the art fromthis detailed description.

Mutant RB69 Polymerases

In some embodiments, provided herein are mutant RB69 DNA polymeraseswhich are mutated versions of a wild-type RB69 polymerase comprising theamino acid sequence of SEQ ID NO: 1 as shown in FIG. 1. These mutant DNApolymerases offer various advantages not provided by other DNApolymerases. In some embodiments, the mutant polymerases of the presentdisclosure exhibit altered nucleotide binding and/or nucleotideincorporation behavior compared to wild type RB69 polymerase. Forexample, in some embodiments the mutant polymerases can exhibitincreased affinity or duration of nucleotide binding in the presence oflabeled nucleotides. In some embodiments, the mutant polymerases canexhibit increased ability to incorporate labeled nucleotides and/orterminator nucleotides.

The mutant polymerases provided herein can be used, for example, incertain single molecule DNA sequencing methods which encompass twosteps: a nucleotide binding step, and/or a nucleotide incorporationstep. Some single molecule sequencing methods require transient bindingof the nucleotide to the polymerase (see e.g., Vander Horn, et al., U.S.Ser. No. 61/184,774; 61/242,763; 61/295,533; and “Mutant DNAPolymerases”, concurrently filed with this application on May 28, 2010,by P. Vander Horn, et al.).

In the transient-binding step, an incoming nucleotide (e.g., labelednucleotide) binds with the polymerase under conditions which inhibitincorporation of the bound nucleotide, and the identity of thetransiently-bound nucleotide is determined. In the second step, a singlenucleotide is incorporated (e.g., terminator nucleotide) so as totranslocate the polymerase to the next position on the DNA template.

The mutant DNA polymerases provided herein can be used for the firstand/or second steps and can provide improved performance in the firstand/or second steps relative to wild-type RB69 polymerase. In certainembodiments, the labeled nucleotide in step one can be replaced with aterminator nucleotide in step two.

In some embodiments, the mutant polymerases can be mesophilicpolymerases. For example, the mesophilic polymerases cantransiently-bind a nucleotide and/or catalyze nucleotide polymerizationat moderate temperatures. The moderate temperatures can range from about15-40° C., or about 15-30° C., or about 15-25° C.

In some embodiments, the mutant polymerases can selectively bind anincoming nucleotide which is complementary to thetemplate-based-nucleotide to form base-pairing interactions.

Typically, the complementary incoming nucleotides andtemplate-based-nucleotides used in the methods of the present disclosurecan form hydrogen bonds by Watson-Crick or Hoogstein binding to form aduplex nucleic acid structure. The complementary base pairing can be thestandard A-T or C-G base pairing, or can be other forms of base-pairinginteractions.

In some embodiments and under suitable conditions, the mutantpolymerases can transiently-bind a nucleotide, without incorporating thebound nucleotide. The suitable conditions include transiently-binding anucleotide to the mutant polymerase in the presence of any combinationof: (1) reduced levels or omission of a metal cation that permitsnucleotide incorporation (e.g., manganese and/or magnesium) and/oraddition of a cation that inhibits nucleotide incorporation (e.g.,calcium); and/or (2) the transiently-bound nucleotide is anon-incorporatable nucleotide. In some embodiments, the mutantpolymerases can transiently bind to a nucleotide with higher affinityand/or for longer duration than a wild type RB69 polymerase comprisingthe amino acid sequence of SEQ ID NO: 1.

In some embodiments, the mutant polymerases can transiently-bind anucleotide which is unlabeled, or a nucleotide which is operably linkedto a reporter moiety. In some embodiments, the mutant polymerases cantransiently-bind to a labeled nucleotide with higher affinity and/or forlonger duration than a wild type RB69 polymerase comprising the aminoacid sequence of SEQ ID NO: 1. An exemplary assay for transient-bindingto a labeled nucleotide is provided herein in Example 2; however, anyother suitable assay can also be employed.

In some embodiments, the mutant polymerases can exhibit an enhancedability to incorporate nucleotides, such as terminator nucleotides(Gardner and Jack 1999 Nucleic Acids Research 27:2545-2555; Gardner andJack 2002 Nucleic Acids Research 30:605-613), includingdideoxynucleotides, acyclo-nucleotides, 3′ mutant nucleotides including3′ azido-mutant nucleotides, and/or ribonucleotides. In someembodiments, the mutant polymerases can incorporate a terminatornucleotide with higher efficiency than a wild type RB69 polymerasecomprising the amino acid sequence of SEQ ID NO: 1. An exemplary assayfor incorporation of a labeled nucleotide is provided herein in Example2; however, any other suitable assay can also be employed.

The mutant polymerases are useful in methods involving successivenucleotide incorporation events, such as DNA sequencing. The mutantpolymerase can be used for partial ribosubstitution (WF Barnes 1978Journal of Molecular Biology 119: 83-99), chain terminating DNAsequencing using dideoxynucleotides (Sanger, Nicklen, and Coulson 1977Proc. Natl. Acad. Sci. USA 74:5463-5467) or acyclo-nucleotides (GLTrainor 1996 U.S. Pat. No. 5,558,991), and/or SNP analysis usingdideoxynucleotides or acyclo-nucleotides (Haff and Simirnov 1997 GenomeMethods 7:378-388).

Isolated Mutant Polymerases

Provided herein are mutant polymerases which include intact subunits,biologically-active fragments, mutant variants, fusion variants,naturally occurring polymerases, or non-naturally occurring polymerases.The mutations can include amino acid substitutions, insertions, ordeletions.

In yet another aspect, the mutant polymerases can be isolated from acell, or generated using recombinant DNA technology or chemicalsynthesis methods. In another aspect, the mutant polymerases can beexpressed in prokaryote, eukaryote, viral, or phage organisms. Inanother aspect, the mutant polymerases can be post-translationallymutant proteins or fragments thereof.

In one aspect, the mutant polymerase can be a recombinant protein whichis produced by a suitable expression vector/host cell system. The mutantpolymerases can be produced by suitable recombinant expression vectorscarrying inserted nucleotide sequences that encode the mutantpolymerases. A nucleic acid molecule encoding the mutant polymerasesequence can be operably linked to a suitable expression vector. Thenucleic acid molecule can be inserted in-frame into the suitableexpression vector. The suitable expression vector, having the insertednucleic acid molecule, can enter a host cell. The suitable expressionvector can replicate in the host cell, such as a phage host, or aprokaryotic or eukaryotic host cell. The suitable expression vector canreplicate autonomously in the host cell, or can be inserted into thehost cell's genome and be replicated as part of the host genome. Thesuitable expression vector can carry a selectable marker that confersresistance to drugs (e.g., kanamycin, ampicillin, tetracycline,chloramphenicol, or the like), or confers a nutrient requirement. Thesuitable expression vector can have one or more restriction sites forinserting the nucleic acid molecule of interest. The suitable expressionvector can include expression control sequences for regulatingtranscription and/or translation of the encoded sequence. The expressioncontrol sequences can include: promoters (e.g., inducible orconstitutive), enhancers, transcription terminators, and secretionsignals. The expression vector can be a plasmid, cosmid, or phagevector. Many expression vectors and suitable host cells are known. Theexpression vector can enter a host cell which can replicate the vector,produce an RNA transcript of the inserted sequence, and/or produceprotein encoded by the inserted sequence. The recombinant mutantpolymerase can include an affinity tag for enrichment or purification,including a poly-amino acid tag (e.g., poly His tag), GST, and/or HAsequence tag.

Methods for Preparing the Mutant Polymerases

Provided herein are methods for preparing the mutant polymerases usingthe suitable recombinant expression vectors and host cells. Preparingrecombinant proteins by expressing the RNA and/or protein encoded by theinserted sequences are well known (Sambrook et al, Molecular Cloning(1989)). For example, the host cell which carries the expression vectorwhich is capable of producing the mutant polymerase, can be cultured ina suitable growth medium (e.g., liquid or semi-solid) having antibioticsfor selection. The host cell can be cultured for a suitable amount oftime (e.g., 8-48 hours), under a suitable temperature range (e.g.,15-40° C.), with or without aeration. The cultured host cells can beharvested, and the mutant polymerases produced in the host cell can beextracted from the host cell. The extracted mutant polymerases can beseparated from non-desirable components of the host cell (e.g., cellwall, DNA, RNA, other proteins) to form an enriched preparation ofmutant DNA polymerases. The enriched preparation can be concentratedusing well-known methods. One exemplary method for isolating a mutantpolymerase of the present disclosure is provided herein in Example 1;however, any other suitable method can be employed.

Selecting Mutant Polymerases

Selecting the mutant polymerases for use in nucleotide transient-bindingand/or the nucleotide incorporation methods can be based on certaindesired polymerase kinetics. For example, the desired polymerasekinetics can be evaluated with respect to any of the following aspectsof polymerase behavior: nucleotide transient-binding (e.g.,association), nucleotide dissociation (intact nucleotide), nucleotidefidelity, nucleotide incorporation (e.g., catalysis), and/or release ofthe cleavage product.

In some embodiments, the mutant polymerases may be selected which retainthe ability to selectively bind complementary nucleotides. In someembodiments, the mutant polymerases may be selected that exhibit amodulated rate (faster or slower) of nucleotide association ordissociation. In some embodiments, the mutant polymerases may beselected that exhibit a reduced rate of nucleotide incorporationactivity (e.g., catalysis) and/or a reduced rate of dissociation of thecleavage product. Examples of other preparing and selecting mutantpolymerases that exhibit nucleotide binding and a reduced rate ofnucleotide incorporation have been described (Rank, U.S. publishedpatent application No. 2008/0108082; Hanzel, U.S. published patentapplication No. 2007/0196846).

In some embodiments, the mutant polymerase can be selected based on thecombination of the mutant polymerase and nucleotides, and the reactionconditions, used to conduct the nucleotide binding and/or nucleotideincorporation reactions.

In some embodiments, the mutant polymerases, nucleotides, and reactionconditions, can be screened for their suitability for use in thenucleotide binding and/or nucleotide incorporation methods, using wellknown screening techniques. For example, the suitable mutant polymerasemay be capable of binding nucleotides and/or incorporating nucleotides.The reaction kinetics for nucleotide binding, association,incorporation, and/or dissociation rates, can be determined using rapidkinetics techniques (e.g., stopped-flow or quench flow techniques).Using stopped-flow or quench flow techniques, the binding kinetics of anucleotide can be estimated by calculating the 1/k_(pol) value.Stopped-flow techniques that analyze absorption and/or fluorescencespectroscopy properties of the nucleotide binding, incorporation, ordissociation rates to a polymerase are well known in the art (Kumar andPatel 1997 Biochemistry 36:13954-13962; Tsai and Johnson 2006Biochemistry 45:9675-9687; Hanzel, U.S. published patent application No.2007/0196846). Other methods include quench flow (Johnson 1986 MethodsEnzymology 134:677-705), time-gated fluorescence decay time measurements(Korlach, U.S. Pat. No. 7,485,424), plate-based assays (Clark, U.S.published patent application No. 2009/0176233), and X-ray crystalstructure analysis (Berman 2007 EMBO Journal 26:3494). Nucleotideincorporation by a mutant polymerase can also be analyzed by gelseparation of the primer extension products. In one embodiment,stopped-flow techniques can be used to screen and select combinations ofnucleotides (including labeled nucleotide analogs) with polymeraseshaving a t_(pol) value (e.g., 1/K_(pol)) which is less than a t⁻¹ (e.g.,1/k_(—1)) value. Stopped-flow techniques for measuring t_(pol) (M PRoettger 2008 Biochemistry 47:9718-9727; M Bakhtina 2009 Biochemistry48:3197-320) and t⁻¹ (M Bakhtina 2009 Biochemistry 48:3197-3208) areknown in the art. An exemplary stopped-flow assay for characterizing theduration of nucleotide-binding to a polymerase is provided herein inExample 2; however, any other suitable assay can also be employed.

In some embodiments, the selection of the mutant polymerase may bedetermined by the level of processivity desired for conductingnucleotide incorporation or polymerization reactions. The mutantpolymerase processivity can be gauged by the number of nucleotidesincorporated for a single binding event between the mutant polymeraseand the target molecule base-paired with the polymerization initiationsite. For example, the processivity level of the mutant polymerase maybe about 1, 5, 10, 20, 25, 50, 100, 250, 500, 750, 1000, 2000, 5000, or10,000 or more nucleotides incorporated with a single binding event(i.e., binding a template molecule). Processivity levels typicallycorrelate with read lengths of a polymerase.

In some embodiments, the selection of the mutant polymerase may bedetermined by the level of fidelity desired, such as the error rate pernucleotide incorporation. The fidelity of a mutant polymerase may bepartly determined by the 3′ →5′ exonuclease activity associated with themutant DNA polymerase. The fidelity of a mutant DNA polymerase may bemeasured using assays well known in the art (Lundburg et al., 1991 Gene,108:1-6). The error rate of the mutant polymerase can be one error perabout 100, or about 250, or about 500, or about 1000, or about 1500incorporated nucleotides. High fidelity polymerases include thoseexhibiting error rates of about 5×10⁻⁶ perbase pair or lower rates.

In some embodiments, the selection of the mutant polymerase may bedetermined by the rate of nucleotide incorporation such as about onenucleotide per 2-5 seconds, or about one nucleotide per second, or about5 nucleotides per second, or about 10 nucleotides per second, or about20 nucleotides per second, or about 30 nucleotides per second, or morethan 40 nucleotides per second, or more than 50-100 per second, or morethan 100 per second. In one embodiment, polymerases exhibiting reducednucleotide incorporation rates include mutant RB69 polymerases havinglysine substituted with leucine, arginine, or other amino acids (Castro2009 Nature Structural and Molecular Biology 16:212-218).

In some embodiments, the mutant polymerase can be a deletion mutant thatretains nucleotide polymerization activity but lacks the 3′ →5′ or 5′→3′ exonuclease activity.

Fusion Proteins

In some embodiments, the mutant polymerase can be a fusion proteincomprising the amino acid sequence of the nucleic acid-dependentpolymerizing enzyme (or a biologically active fragment thereof) operablylinked to the amino acid sequence of a second biologically active enzyme(or a biologically active fragment thereof). The second enzyme sequencemay be linked to the amino or carboxyl end of the mutant polymerasesequence, or may be inserted within the mutant polymerase sequence. Themutant polymerase sequence may be linked to the amino or carboxyl end ofthe second enzyme sequence, or may be inserted within the second enzymesequence. The mutant polymerase and second enzyme sequences can belinked to each other in a manner that does not interfere with polymeraseactivity or with a nucleotide binding to the polymerase or withnucleotide polymerization, or does not interfere with the activity ofthe second enzyme sequence. The fusion protein can include the aminoacid sequences of the mutant polymerase chemically linked to the aminoacid sequence of the second enzyme.

Tagged Polymerases

In some embodiments, the mutant polymerase can be operably linked to alinker moiety includes: a covalent or non-covalent bond; amino acid tag(e.g., poly-amino acid tag, poly-His tag, 6His-tag); chemical compound(e.g., polyethylene glycol); protein-protein binding pair (e.g.,biotin-avidin); affinity coupling; capture probes; or any combination ofthese. The linker moiety can be separate from or part of the mutantpolymerase (e.g., recombinant His-tagged polymerase). Typically, thelinker moiety does not interfere with the nucleotide binding activity,or catalytic activity of the mutant polymerase.

Mutant Polymerases Linked to Reporter Moieties

Provided herein are mutant DNA polymerases which are unlinked, or areoperably linked to reporter moieties via a suitable linker. The reportermoiety can be an energy transfer donor which is capable of FRET with aproximal energy transfer acceptor. The energy transfer donor can be afluorescent dye or nanoparticle. Typically, the reporter moiety and thesuitable linker do not interfere with the function or activity of themutant polymerases.

In some embodiments, the suitable linker can mediate covalent ornon-covalent attachment. Examples of non-covalent attachment includes:ionic, hydrogen bonding, dipole-dipole interactions, van der Waalsinteractions, ionic interactions, and hydrophobic interactions. Inparticular, examples of non-covalent attachment includes: nucleic acidhybridization, protein aptamer-template binding, electrostaticinteraction, hydrophobic interaction, non-specific adsorption, andsolvent evaporation.

In some embodiments, the suitable linker can be rigid or flexible. Therigid linker can be used to improve a FRET signal by optimizing theorientation of the energy transfer dye. Examples of rigid linkersinclude benzyl linkers, proline or poly-proline linkers (S. Flemer, etal., 2008 Journal Org. Chem. 73:7593-7602), bis-azide linkers (M. P. L.Werts, et al., 2003 Macromolecules 36:7004-7013), and rigid linkerssynthesized by modifying the so-called “click” chemistry scheme that isdescribed by Megiatto and Schuster (2008 Journal of the Am. Chem. Soc.130:12872-12873).

The suitable linker can optimize proximity, length, distance,orientation, or charge. For example, the linker can be a cationicpoly-arginine spacer linker or an imidazolium spacer molecule.

In some embodiments, the suitable linker can be a cleavable,self-cleavable, or fragmentable linker. The self-cleaving linker can bea trimethyl lock or a quinone methide linker. The suitable linker can becleaved or fragmented via light (e.g., photo-cleavable linkers), achemical reaction, enzymatic activity, heat, acid, or base.

The suitable linker can be linear, non-linear, branched, bifunctional,trifunctional, homofunctional, or heterofunctional. Some linkers havependant side chains or pendant functional groups, or both. The suitablelinker comprises about 1-100 plural valent atoms. In some embodiments,the linker moiety comprises about 1-40 plural valent atoms, or more,selected from the group consisting of C, N, O, S and P.

Linking the Mutant Polymerases and Nanoparticles

The mutant polymerases can be operably linked to at least onenanoparticle using well known linking schemes. In general, these linkingschemes include: (1) a condensation reaction between the amines on theproteins and the carboxy groups on the nanoparticle using, for example,1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC); (2) directlybinding thiolated proteins to the nanoparticles using dativethiol-bonding between the cysteine residues on the protein and thesulfur atoms on the nanoparticle surface; (3) metal-affinitycoordination between the histidine residues in the proteins and the Znatoms on the nanoparticle surface; or (4) adsorption or non-covalentself-assembly of protein (e.g., mutant polymerase) on to thenanoparticle surface.

In some embodiments, the nanoparticles can have ligand coatings, such ascarboxyl groups (e.g., as carboxyl-derived amphiphilic compounds) whichcan be reacted with the amines, hydrazines, or hydroxylamines on themutant polymerases in a condensation reaction (e.g., using EDC).

The nanoparticle ligands can be amino-derivatized ligands that permitcrosslinking with amine reactive groups such as isothiocyanates,succinimidyl esters and other active esters.

The mutant polymerases can be attached directly to the nanoparticle. Themutant polymerases having a poly-His sequence (e.g., tag) can beattached directly to a nanoparticle via metal-affinity coordinationbetween the nanoparticle Zn atoms (e.g., on the shell) and histidineresidues. The histidine residues also have varying affinities for othermetals including Ni²⁺, Co²⁺, Cu²⁺, Mn²⁺, Fe²⁺ and Fe³⁺. The mutantpolymerases can be attached directly to the nanoparticle via dativethiol bonding between S atoms in the nanoparticle and cysteine residueson the surface or in the protein.

In some embodiments, the mutant polymerases can adsorb (e.g.,non-covalently) on to the nanoparticle. For example, positively chargedpolymerases can adsorb on to the negatively charged a nanoparticle.

The nanoparticle ligand coating can be PEG or biotin, which can belinked to the mutant polymerase having an avidin-like molecule, viaamine linking chemistry. For example, the PEG coating can be reactedwith a cross-linker (e.g., bis(sulfosuccinimidyl) suberate; (BS3)) foramine linking with streptavidin.

In some embodiments, the nanoparticle can be reacted with a compoundwhich can bind to some or all of the binding sites on the nanoparticleshell in order to mask the binding sites. The compounds includehorseradish peroxidase (HRP), glutathione 5-transferase (GST),uracil-DNA glycosylase (UDG), uracil glycosylase inhibitor (UGI), orbovine serum albumin (BSA). For example, a non-masked nanoparticle maybe capable of binding many protein molecules. In another example apartially masked nanoparticle, which is coated with HRP, GST, UDG, UGI,and/or BSA, may be capable of binding fewer protein molecules. In oneembodiment, one nanoparticle can bind about 1-5, or about 5-10, or about10-20, or about 20-50, or more mutant polymerases. In one embodiment,one nanoparticle can bind one mutant polymerase.

In one embodiment, the nanoparticle can be reacted to carry aniodoacetal functional group which can bind to a mutant polymerasecarrying a phosphorothioate functional group on a recombinantlyintroduced serine residue.

Dispersibility of Nanoparticles

The nanoparticles can be reacted with compounds that alter thedispersibility properties of the nanoparticles, or form reactive groupsfor covalent or non-covalent interactions with the mutant polymerases.

In one aspect, the nanoparticles can be modified to permitdispersibility in a solvent. For example, the nanoparticles can bedispersible in aqueous solvents, including: water, and aqueous solutionsand buffers. Nanoparticles that are not dispersible in aqueous solventstypically have hydrophobic ligands which are intimately associated withthe outer shell. The ligands, which are sometimes referred to as a cap,can be trioctylphosphine (TOP), trioctylphosphine oxide (TOPO), oleicacid, or tetradecylphosphonic acid (TDPA). The aqueous-dispersiblenanoparticles can be modified to have hydrophilic ligands via a capexchange procedure. The cap exchange procedure can involve substitutingthe exposed groups on the nanoparticle (e.g., hydrophobic caps) withheterofunctional ligands. The nanoparticle surface can be modified tohave hydrophilic ligands by encapsulating the nanoparticle in a coatingof heterofunctional ligands. The heterofunctional ligands can include athiol anchor moiety and a hydrophilic moiety (e.g., hydroxyl orcarboxyl). Examples of heterofunctional ligands include thiol andphosphine mono and multidentate ligands, such as: mercaptocarbonicacids; alkythiol terminated molecules, thioalkylated molecules, anddihydrolipoic acid derivatives. Another example involves forming apolymerized silica shell on the nanoparticle surface. The silica shellcan be functionalized with polar groups using, for example,mercaptopropyl silanols or amine box dendrimers. In yet another example,the native functional groups on the nanoparticle surface are preserved.The nanoparticles are reacted with amphiphilic diblock or triblockcopolymers, or phospholipids, which have hydrophobic groups thatinterdigitate with the native functional groups on the nanoparticleshell. The amphiphilic copolymers and have hydrophilic groups thatpermit aqueous dispersal. The interdigitating compounds include:phosphatidylethanol amine, phosphatidycholine micelles, modified acrylicacid polymers, poly (maleic anhydride)-alt-1-tetradecene, amphiphilictriblock copolymer (Gao 2004 Nature Biotechnology 22:969-976), andamphiphilic saccharides. Another procedure for preserving thenanoparticle native functional groups involves reacting the nanoparticlewith oligomeric phosphines that carry hydrophilic functional groupsand/or carry a protein-protein binding partner (e.g., avidin orderivative thereof). Proteins can also be nonspecifically adsorbed on tothe nanoparticle surface.

Mutant RB69 Polymerase Sequences

In some embodiments, the mutant RB69 polymerases of the presentdisclosure can exhibit reduced exonuclease activity. For example, themutant polymerase can include mutations at (in single letter amino acidcode): D222A, D327A, or D222A/D327A (numbering is based on the aminoacid sequence shown in SEQ ID NOS:1 or 2).

In some embodiments, the mutant RB69 polymerases of the presentdisclosure can exhibit an enhanced ability to incorporate nucleotides(Gardner and Jack 1999 Nucleic Acids Research 27:2545-2555; Gardner andJack 2002 Nucleic Acids Research 30:605-613), includingdideoxynucleotides (N558L, Y416A, or N558L/Y416A), acyclo-nucleotides(N558L), 3′ modified nucleotides including 3′ azido-modified nucleotides(LYP amino acid motif at positions 415-417 mutated to SAV), andribonucleotides (Y416A, N558L/Y416A), and other modifications (N558L, orLYP amino acid motif at positions 415-417 mutated to SAV) (numbering isbased on the amino acid sequence shown in SEQ ID NO:1).

In some embodiments, the mutant RB69 polymerase can include any one ormore mutations selected from the group consisting of: D222A; D327A;L415S; Y416A; P417V; N558L; D22A/D327A; D222A/D327A/N558L;D22A/D327A/Y416A; D222A/D327A/N558L/Y416A;D222A/D327A/L415S/Y416A/P417V; and D222A/D327A/N558L/L415S/Y416A/P417V,wherein the numbering is based on the amino acid sequence shown in SEQID NO:1).

For example, the mutant RB69 polymerases can include any one of themutant polymerases listed in Table 1 below.

TABLE 1 WT 6HIS-tag SX 6HIS-tag D327A DX D222A, D327A FDX D222A, N558LD327A PDX D222A, Y416A D327A FPDX D222A, N558L Y416A D327A 3PDX D222A,415LYP > SAV D327A F3PDX D222A, N558L 415LYP > SAV D327A

Also provided herein are any nucleotide sequences that encode the mutantRB69 polymerases of the present disclosure. It is well known in the artthat the genetic code is degenerate. Accordingly, more than one tripletcodon can encode the same amino acid. Table 2 below illustrates thedegenerate genetic code.

TABLE 2 Amino Acid Symbol Symbol Codons Alanine Ala A GCU, GCC, GCA, GCGCysteine Cys C UGU, UGC Aspartic Acid Asp D GAU, GAC Glutamic Acid Glu EGAA, GAG Phenylalanine Phe F UUU, UUC Glycine Gly G GGU, GGC, GGA, GGGHistidine His H CAU, CAC Isoleucine Ile I AUU, AUC, AUA Lysine Lys KAAA, AAG Leucine Leu L UUA, UUG, CUU, CUC, CUA, CUG Methionine Met M AUGAsparagine Asn N AAU, AAC Proline Pro P CCU, CCC, CCA, CCG Glutamine GlnQ CAA, CAG Arginine Arg R CGU, CGC, CGA, CGG, AGA, AGG Serine Ser S UCU,UCC, UCA, UCG, AGU, AGC Threonine Thr T ACU, ACC, ACA, ACG Valine Val VGUU, GUC, GUA, GUG Tryptophan Trp W UGG Tyrosine Tyr Y UAU, UAC

In one embodiment, the mutant DNA polymerases comprise amino acidsequences of about 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to anyone of the amino acid sequences of SEQ ID NOS:2-8. In some embodiments,these mutant DNA polymerases can exhibit altered, e.g., increased ordecreased, ability to transiently-bind a labeled nucleotide relative tothe binding capability of a wild type RB69 polymerase according to SEQID NO: 1. In some embodiments, these mutant DNA polymerases can exhibitaltered, e.g., increased or decreased, ability to incorporate anucleotide (e.g., a labeled nucleotide or terminator nucleotide)relative to a wild type RB69 polymerase having the amino acid sequencingof SEQ ID NO: 1.

In another embodiment, the mutant DNA polymerase can exhibit exonucleaseactivity. In some embodiments, the mutant DNA polymerase can exhibitreduced exonuclease activity relative to the RB69 polymerase having theamino acid sequence of SEQ ID NO: 1. For example, the mutant DNApolymerases can exhibit exonuclease activity, and comprise an asparticacid (D) at position 222 and/or an aspartic acid (D) at position 327 asshown in any one of SEQ ID NO:1-8. In another example, the mutant DNApolymerase can exhibit reduced exonuclease activity relative to the wildtype RB69 polymerase having the amino acid sequence of SEQ ID NO: 1, andcan further comprise an alanine (A) at position 222 and/or an alanine(A) at position 327 as shown in any one of SEQ ID NO:1-8. Optionally,the mutant DNA polymerase can comprise further modifications (e.g.,amino acid substitution(s)) at any other position of any one of SEQ IDNO:1-8.

In some embodiments, the mutant polymerase comprises the amino acidsequence that is at least 80%, 85%, 90%, 95%, 97% or 99% identical toany amino acid sequence selected from the group consisting of: SEQ IDNO: 1, SEQ ID NO: 2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6,SEQ ID NO:7, and SEQ ID NO:8. Optionally, the mutant polymerase canexhibit increased duration of binding to a labeled nucleotide relativeto a wild type RB69 polymerase having the amino acid. In someembodiments, the duration of nucleotide binding can be estimated using astopped-flow assay as described, for example, in Example 2;alternatively, any other suitable assay for measuring the duration ofnucleotide binding can be used. Optionally, the mutant polymerase canexhibit increased nucleotide incorporation activity for a labelednucleotide and/or for a terminator nucleotide. In some embodiments, thenucleotide incorporation activity can be measured using the assayprocedure described in Example 3; alternatively, any other suitableassay for measuring nucleotide incorporation activity can be used.

Nucleotides

The mutant DNA polymerases can transiently-bind and/or incorporate anucleotide. In one aspect, nucleotides are compounds that can bindselectively to, or can be incorporated by, the mutant polymerasesprovided herein. Such nucleotides include not only naturally-occurringnucleotides but also any analogs, regardless of their structure, thatcan bind selectively to, or can be polymerized by, the mutantpolymerase. While naturally-occurring nucleotides typically comprisebase, sugar and phosphate moieties, the nucleotides of the presentdisclosure can include compounds lacking any one, some or all of suchmoieties.

In some embodiments, the nucleotides can optionally include a chain ofphosphorus atoms comprising three, four, five, six, seven, eight, nine,ten or more phosphorus atoms. In some embodiments, the phosphorus chaincan be attached to any carbon of a sugar ring, such as the 5′ carbon.The phosphorus chain can be linked to the sugar with an intervening O orS. In one embodiment, one or more phosphorus atoms in the chain can bepart of a phosphate group having P and O. In another embodiment, thephosphorus atoms in the chain can be linked together with intervening O,NH, S, methylene, substituted methylene, ethylene, substituted ethylene,CNH₂, C(O), C(CH₂), CH₂CH₂, or C(OH)CH₂R (where R can be a 4-pyridine or1-imidazole). In one embodiment, the phosphorus atoms in the chain canhave side groups having O, BH₃, or S. In the phosphorus chain, aphosphorus atom with a side group other than O can be a substitutedphosphate group. Some examples of nucleotides are described in Xu, U.S.Pat. No. 7,405,281. Some examples of nucleotides that can be used in thedisclosed methods and compositions include, but are not limited to,ribonucleotides, deoxyribonucleotides, modified ribonucleotides,modified deoxyribonucleotides, ribonucleotide polyphosphates,deoxyribonucleotide polyphosphates, modified ribonucleotidepolyphosphates, modified deoxyribonucleotide polyphosphates, peptidenucleotides, metallonucleosides, phosphonate nucleosides, and modifiedphosphate-sugar backbone nucleotides, analogs, derivatives, or variantsof the foregoing compounds, and the like. In some embodiments, thenucleotide can comprise non-oxygen moieties such as, for example, thio-or borano-moieties, in place of the oxygen moiety bridging the alphaphosphate and the sugar of the nucleotide, or the alpha and betaphosphates of the nucleotide, or the beta and gamma phosphates of thenucleotide, or between any other two phosphates of the nucleotide, orany combination thereof.

In some embodiments, the nucleotides can be operably linked to areporter moiety (e.g., labeled nucleotides) or can be un-labeled. Thereporter moiety can be operably linked to any part of the nucleotide,including the base, sugar or to any phosphate group or substitutephosphate group (e.g., a terminal phosphate group, or any otherphosphate group that is released from the nucleotide duringincorporation by the polymerase).

Typically, the reporter moiety generates, or causes to be generated, adetectable signal. Any suitable reporter moiety may be used, includingluminescent, photoluminescent, electroluminescent, bioluminescent,chemiluminescent, fluorescent, phosphorescent, chromophore,radioisotope, electrochemical, mass spectrometry, Raman, hapten,affinity tag, atom, or an enzyme. The reporter moiety generates adetectable signal resulting from a chemical or physical change (e.g.,heat, light, electrical, pH, salt concentration, enzymatic activity, orproximity events). A proximity event includes two reporter moietiesapproaching each other, or associating with each other, or binding eachother. For example, proximity of an energy transfer donor and acceptormoiety can occur when an acceptor-labeled nucleotide binds the activesite of a donor-labeled mutant polymerase, resulting in energy transferfrom the donor to the acceptor. In one embodiment, energy transferdistances (e.g., FRET distances) can be about 5-20 nm. The reportermoieties may be selected so that each absorbs excitation radiationand/or emits fluorescence at a wavelength distinguishable from the otherreporter moieties to permit monitoring the presence of differentreporter moieties in the same reaction. Two or more different reportermoieties can be selected having spectrally distinct emission profiles,or having minimal overlapping spectral emission profiles.

The fluorescent moiety includes: rhodols; resorufins; coumarins;xanthenes; acridines; fluoresceins; rhodamines; erythrins; cyanins;phthalaldehydes; naphthylamines; fluorescamines; benzoxadiazoles;stilbenes; pyrenes; indoles; borapolyazaindacenes; quinazolinones;eosin; erythrosin; and Malachite green.

Terminator Nucleotides

In some embodiments, the mutant DNA polymerases can transiently bindand/or incorporate a terminator nucleotide. Optionally, the terminatornucleotides can be incorporated in a template-dependent manner by themutant polymerase. In some embodiments, the incorporated terminatornucleotide can inhibit the subsequent incorporation of anothernucleotide by the polymerase.

In some embodiments, the terminator nucleotide comprises a nucleotideoperably linked to an inhibitor moiety. Typically, the inhibitor moietycan comprise any chemical compound or chemical group which permitsincorporation of the terminator nucleotide by the mutant polymerase butinhibits incorporation of the next nucleotide. Thus, the mutantpolymerase can incorporate one and only one terminator nucleotide,thereby advancing nucleotide incorporation by only one base. Theinhibitor moiety can be operably linked to any portion of the nucleosideor nucleotide (e.g., any phosphate group, or base or sugar moiety). Thesame type or different types of inhibitor moieties can be operablylinked to different types of nucleotides. The terminator nucleotide canbe resistant to degradation by 3′-5′ exonuclease activity of the mutantpolymerase.

On the terminator nucleotide, the inhibitor moiety can be modifiedfollowing incorporation of the terminator nucleotide so that the nextnucleotide can be incorporated (e.g., reversible terminator nucleotide).Alternatively, the inhibitor moiety can be cleaved off or otherwiseremoved following incorporation (de-blocking) to permit incorporation ofthe next nucleotide.

In one embodiment, the terminator nucleotides can be non-labeled, or canbe operably linked to at least one reporter moiety at any position ofthe base or sugar, or any of the phosphate groups (alpha, beta, gamma,or terminal phosphate group).

Many suitable terminator nucleotides having inhibitor moieties attachedto the sugar 3′ position, base-linked dyes, where the linkers arecleavable under the same conditions are described by Tsien(WO/1991/006678). Additionally, suitable terminator nucleotides havingphotocleavable linkers are described by Stemple (U.S. Pat. No.7,270,951; Turner, U.S. Pat. No. 7,476,504).

Inhibitor Moieties

The terminator nucleotides can optionally comprise a nucleotide operablylinked to at least one suitable inhibitor moiety. In some embodiments,the inhibitor moiety comprises any chemical compound or chemical groupwhich permits polymerization, in a template-dependent manner, by themutant polymerase, but inhibits incorporation of the next nucleotide.The inhibitor moiety can modify, substitute, or protect, any portion ofthe nucleotide (e.g., base, sugar, or phosphate group). A suitableinhibitor moiety can be operably linked to any part of the nucleotide(or nucleoside) including the base or sugar moiety, or any phosphategroup, or any analogous structure corresponding to any of thesemoieties. In some embodiments, the suitable inhibitor moiety can permitincorporation of the terminator nucleotide, in a polymerase-driven,template-dependent manner, but inhibits, stalls, or slows downincorporation of the next nucleotide by the mutant polymerase. In someembodiments, the suitable inhibitor moiety inhibits incorporation of thenext nucleotide by physical, chemical, or charge interaction with themutant polymerase and/or incoming nucleotide.

The suitable inhibitor moiety can be operably linked to the 2′ or 3′position of the sugar moiety. In one embodiment, the 2′ or 3′-H or —OHgroup of the sugar moiety can be modified, substituted, or protected.For example, it is well known that DNA polymerases can require apolymerization initiation site having a terminal 3′-OH group. Thus, theinhibitor moiety can be any chemical group or compound, which is not an—OH group, operably linked to the 3′ C of the sugar moiety or analogousstructure within the nucleotide. In some embodiments, the suitableinhibitor moiety can be an —H group operably linked to the 3′ C of thesugar moiety or analogous structure within the nucleotide. Suchembodiments include dideoxynucleosides and dideoxynucleotides. Examplesof inhibitor moieties attached to the sugar 3′ position are described byBalasubramanian (U.S. Pat. No. 7,427,673) and Milton (U.S. Pat. No.7,541,444).

The suitable inhibitor moiety can be operably linked to any position ofthe nitrogenous base, such as a purine group or analogous structurewithin the nucleotide, including the C2, C4, C5, N3, or C6, of cytosine,thymine, and uracil, or to any analogous structure in the nucleotide.The suitable inhibitor moiety can be operably linked to any position ofthe pyrimidine group or analogous structure within the nucleotide,including the C2, C6, C8, N3 and N7 of adenine and guanine.

The suitable inhibitor moiety can be operably linked to any phosphategroup or analogous structure within the nucleotide, such as the alpha,beta or gamma phosphate group, the terminal phosphate group, or anyother phosphate group that is released upon incorporation.

In some embodiments, the suitable inhibitor moiety can be linked to anyportion of the nucleoside or nucleotide, and sterically hinder theincoming nucleotide. In some embodiments, the suitable inhibitor moietycan be a charged group (positive or negative) and linked to any portionof the nucleoside or nucleotide and can inhibit the polymerase fromincorporating the next nucleotide. In some embodiments, the suitableinhibitor moiety can be linked to at least one of: asterically-hindering group, fluorophore, and/or quencher, in any orderand in any combination.

In some embodiments, the suitable inhibitor moiety comprises any groupincluding: amine, alkyl, alkenyl, alkynyl, alkyl amide, aryl, ether,ester, benzyl, propargyl, propynyl, phosphate, or analog thereof. Forexample, the suitable inhibitor moiety can be a 3′-O-allyl moiety(Ruparel, et al., 2005 Proc. Natl. Acad. Sci. USA 102:5932-5937).

Suitable inhibitor moieties are well known in the art, and include:fluorenylmethyloxycarbonyl (FMOC), 4-(anisyl)diphenylmethyltrityl(MMTr), dimethoxytrityl (DMTr), monomethoxytrityl, trityl (Tr), benzoyl(Bz), isobutyryl (ib), pixyl (pi), ter-butyl-dimethylsilyl (TBMS), and1-(2-fluorophenyl)-4-methoxypiperidin 4-yl (FPMP). See also T W Greene1981, in “Protective Groups in Organic Synthesis”, publishersWiley-Interscience; Beaucage and Iyer 1992 Tetrahedron, 48:2223-2311;Beaucage and Iyer 1993 Tetrahedron 49:10441-10488; and Scaringe et al.,1998 J. Am. Chem. Soc. 120:11820-11821.

In some embodiments, the suitable inhibitor moiety can be a reportermoiety (e.g., fluorescent dye) operably linked to the base or sugarmoiety. For example, a fluorescent dye operably linked to the base via a2-nitrobenzyl group, where the 2-nitrobenyl group has the alpha carbonsubstituted with one alkyl or aryl group (Wu, et al., U.S. publishedpatent application No. 2008/0132692). The 2-nitrobenzyl group can bephotocleavable.

In another example, the suitable inhibitor moiety can be a reportermoiety (e.g., fluorescent dye, e.g., ALEXA FLUOR 549) operably linked tothe 5 position of pyrimidines or the 7 position of the purines, via acleavable disulfide linker (Turcatti, et al., 2008 Nucleic AcidsResearch vol. 36, No. 4, doi:10.1093/nar/gkn021).

In yet another example, the suitable inhibitor moiety can be arhodamine-type dyes, such as R6G, R110, ROX, or TAMRA, ordichloro-derivatives thereof, which are based-linked dyes, including thecommercially-available rhodamine dye terminator nucleotides from AppliedBiosystems.

In some embodiments, the suitable inhibitor moiety can be a chargedgroup (positive or negative) or a group capable of becoming charged(Efcavitch, U.S. published patent application No. 2009/0061437),including a carboxylic acid, carboxylated, phosphate, di-phosphate,peptide, dipeptide, sulfate, disulfate, caproic acid, or amino acid(e.g., a negatively charged amino acid such as aspartic acid, glutamicacid, histidine, lysine, or arginine).

In some embodiments, the suitable inhibitor moiety can be anon-incorporatable nucleotide or nucleoside which is linked to the baseby a tether. The tether can be linked to a fluorescent label. The tethercan include a cleavable moiety, such as a disulfide group (Siddiqi, U.S.published patent application No. 2008/0103053 and 2008/0227970).

In some embodiments, the suitable inhibitor moiety can be ahydrocaryldithiomethyl-modified compound (Kwiatkowski, U.S. Pat. No.7,279,563.

The suitable inhibitor moiety can include an ethyl dithio linker(Siddiqi, U.S. published patent application No. 2008/0269476).

In some embodiments, the suitable inhibitor moiety can be an alkyl chainhomologue having any chain length, which can be produced by replacing2-bromoethanol and ethylsulfide reagents with any alkyl chain homologue(Siddiqi, U.S. published patent application No. 2008/0269476).

In some embodiments, the suitable inhibitor moiety can be any phosphate,SO₃, or C(O)R group, or modified groups thereof (Lee, U.S. publishedpatent application No. 2008/0050780). In the C(O)R group, R can be an H,alkyl, benzyl, aryl, alkenyl, alkynyl group, any combination thereof.

In one embodiment, removal or modification of the inhibitor moiety whichis attached to the 3′ C of the sugar moiety, and restoration of a 3′-OHgroup, can permit incorporation of a subsequent nucleotide (e.g.,reversible terminator nucleotide). In another embodiment, removal ormodification of the inhibitor moiety which is attached to the sugar,base, or phosphate group, can permit incorporation of a subsequentnucleotide (e.g., reversible terminator nucleotide).

Linkers for Terminator Nucleotides

In some embodiments, the terminator nucleotide is operably linked to theinhibitor moiety via a suitable linker. Typically, the suitable linkerdoes not interfere with the function or activity of the nucleotide,nucleoside, or inhibitor moiety. The suitable linker can be cleavable orfragmentable to permit removal of the inhibitor moiety. The suitablelinker can be the inhibitor moiety. In one embodiment, the nucleotidecan be attached directly to the inhibitor moiety without an interveninglinker. Various linkers and linker chemistries for generating theterminator nucleotides are disclosed infra.

Optionally, the terminator nucleotides can be linked to inhibitormoieties using any suitable linking scheme, including linking schemesusing amine linkers (Hobbs, U.S. Pat. No. 5,151,507), or primary orsecondary amines, or a rigid hydrocarbon arm (RF Service, 1998 Science282:1020-21).

Optionally, the terminator nucleotide can include more than one linker,where the linkers are the same or different. The multiple linkers can beremoved, cleaved or fragmented using different temperatures, enzymaticactivities, chemical agents, and/or different wavelengths ofelectromagnetic radiation.

Cleavable Linkers

In some embodiments, the linker that links the inhibitor moiety to theterminator nucleotide can be cleavable by heat, enzymatic activity,chemical agent, or electromagnetic radiation. Cleavable groups include:disulfide, amide, thioamide, ester, thioester, vicinal diol, orhemiacetal. Other cleavable bonds include enzymatically-cleavable bonds,such as peptide bonds (cleaved by peptidases), phosphate bonds (cleavedby phosphatases), nucleic acid bonds (cleaved by endonucleases), andsugar bonds (cleaved by glycosidases).

In one embodiment, the cleavable linker can be a photocleavable linker,such as a 2-nitrobenzyl linker (Bai 2004 Nucl. Acid Res. 32:535-541;Seo, et al., 2005 Proc. Natl. Acad. Sci. USA 102:5926-5931; Wu, et al.,2007 Proc. Natl. Acad. Sci. USA 104:16462-16467), or others (Lyle, U.S.published patent application No. 2008/0009007). Analogs of the2-nitrobenzyl linker, and other photocleavable linkers can be used ascleavable blocking groups, including: 2-nitrobenzyloxycarbonyl (NBOC);nitroveratryl; 1-pyrenylmethyl; 6-nitroveratryloxycarbonyl (NVOC);dimethyldimethoxy-benzyloxycarbonyl (DDZ); 5-bromo-7-nitroindolinyl;O-hydroxy-alpha-methyl-cinnamoyl; methyl-6-nitroveratryloxycarbonyl;methyl-6-nitropiperonyloxycarbonyl; 2-oxymethylene anthraquinone;dimethoxybenzyloxy carbonyl; 5-bromo-7-nitroindolinyl;O-hydroxy-alpha-methyl cinnamoyl; t-butyl oxycarbonyl (TBOC), and2-oxymethylene anthriquinone (see: McGall, U.S. Pat. No. 5,412,087;Pirrung, U.S. Pat. No. 5,143,854; and Conrad, U.S. Pat. No. 5,773,308).The photocleavable linkers can be illuminated with an electromagneticsource at about 320-800 nm, depending on the particular linker, toachieve cleavage. For example, 1-(2-nitrophenyl)ethyl can be cleavedwith light at about 300-350 nm, and 5-bromo-7-nitroindolinyl can becleaved with light at about 420 nm. In another embodiment, thephotocleavable linker can serve as the inhibitor moiety.

In another embodiment, the terminator nucleotide can include two or morecleavable linkers, each attached to a different portion of thenucleotide. For example, the terminator nucleotide can include twodifferent photo-cleavable linkers that are cleavable with the same ordifferent wavelengths of light.

In another embodiment, the linker can be an ethyl dithio or an alkylchain linker (Siddiqi, U.S. published patent application Nos.2008/0269476 and 2008/0286837). In another embodiment, the cleavablelinker can be a disulfide-linker which is a chemically-cleavable linker(Shimkus 1985 Proc. Natl. Acad. Sci. USA 82:2593-2597). In yet anotherembodiment, the cleavable linker can be an allyl moiety which iscleavable by palladium (Pd(0)) in a deallylation reaction (Ju, et al.,2006 Proc. Natl. Acad. Sci. USA 103:19635-19640; Wu, et al., 2007 Proc.Natl. Acad. Sci. USA 104:16462-16467), or an azidomethyl group which iscleavable with Tris(2-carboxyethyl)phosphine (TCEP) in aqueous solution(Guo, et al., 2008 Proc. Natl. Acad. Sci. USA 105:9145-9150; Bentley, etal., 2008 Nature 456:53-59, and Supplemental Materials and Methods). Instill another embodiment, the linker can be cleavable with silvernitrate (AgNO₃). In another embodiment, an azidomethyl group can serveas an inhibitor moiety and a cleavable linker.

A procedure for synthesizing a terminator nucleotide having an unblocked3′OH group and carrying a biotin molecule linked to the base moiety(N6-alkylated base) via a 2-nitrobenzyl linker may be adapted from themethod described by Wu (Nucl. Acid. Res. 2007, 35:6339-6349).

Fragmentable Linkers

In some embodiments, the inhibitor moiety is linked to the terminatornucleotide via a suitable fragmentable linker. Optionally, the suitablefragmentable linker is capable of fragmenting in an electronic cascadeself-elimination reaction (Graham, U.S. published patent application No.2006/0003383; and Lee, U.S. published patent application No.2008/0050780). In some embodiments, the fragmentable linker comprises atrigger moiety. The trigger moiety comprises a substrate that can becleaved or “activated” by a specified trigger agent. Activation of thetrigger moiety initiates a spontaneous rearrangement that results in thefragmentation of the linker and release of the enjoined compound. Forexample, the trigger moiety can initiate a ring closure mechanism orelimination reaction. Various elimination reactions, include 1,4-, 1,6-and 1,8-elimination reactions.

Any means of activating the trigger moiety may be used. Selection of aparticular means of activation, and hence the trigger moiety, maydepend, in part, on the particular fragmentation reaction desired. Insome embodiments, activation is based upon cleavage of the triggermoiety. The trigger moiety can include a cleavage site that is cleavableby a chemical reagent or enzyme. For example, the trigger moiety caninclude a cleavage recognition site that is cleavable by a sulfatase(e.g., SO₃ and analogs thereof), esterase, phosphatase, nuclease,glycosidase, lipase, esterase, protease, or catalytic antibody.

Non-Incorporatable Nucleotides

In some embodiments, the mutant DNA polymerases can transiently-bind anon-incorporatable nucleotide. The non-incorporatable nucleotides may ormay not have a structure similar to that of a native nucleotide whichmay include base, sugar, and phosphate moieties.

Optionally, the non-incorporatable nucleotides can bind thepolymerase/template complex in a template-dependent manner, or can actas a universal mimetic and bind the polymerase/template complex in anon-template-dependent manner. The non-incorporatable nucleotides can bea nucleotide mimetic of incorporatable nucleotides, such as adenosine,guanosine, cytidine, thymidine or uridine nucleotides. Thenon-incorporatable nucleotide includes any compound having a nucleotidestructure, or a portion thereof, which can bind a mutant polymerase.

For example, the non-incorporatable nucleotides can have the generalstructure:

R₁₁—(—P)_(n)—S—B

Where B can be a base moiety, such as a hetero cyclic base whichincludes substituted or unsubstituted nitrogen-containing heteroaromaticring. Where S can be a sugar moiety, such as a ribosyl, riboxyl, orglucosyl group. Where n can be 1-10, or more. Where P can be one or moresubstituted or unsubstituted phosphate or phosphonate groups. Where R₁₁,if included, can be a reporter moiety (e.g., a fluorescent dye). In oneembodiment, the non-incorporatable nucleotide having multiple phosphateor phosphonate groups, the linkage between the phosphate or phosphonategroups can be non-hydrolyzable by the mutant polymerase. Thenon-hydrolyzable linkages include, but are not limited to, amino, alkyl,methyl, and thio groups. Non-incorporatable nucleotide tetraphosphateanalogs having alpha-thio or alpha boreno substitutions having beendescribed (Rank, U.S. published patent application No. 2008/0108082; andGelfand, U.S. published patent application No. 2008/0293071).

Optionally, the phosphate or phosphonate portion of thenon-incorporatable nucleotide can have the general structure:

Where B can be a base moiety and S can be a sugar moiety. Where any oneof the R₁-R₇ groups can render the nucleotide non-hydrolyzable by apolymerase (e.g., the mutant polymerase). Where the sugar C5 positioncan be CH₂, CH₂O, CH═, CHR, or CH₂. Where the R₁ group can be O, S, CH═,CH(CN), or NH. Where the R₂, R₃, and R₄, groups can independently be O,BH₃, or SH. Where the R₅ and R₆ groups can independently be an amino,alkyl, methyl, thio group, or CHF, CF₂, CHBr, CCl₂, O—O, or —C≡C—. Wherethe R₇ group can be oxygen, or one or more additional phosphate orphosphonate groups, or can be a reporter moiety. Where R₈ can be SH,BH₃, CH₃, NH₂, or a phenyl group or phenyl ring. Where R₉ can be SH.Where R₁₀ can be CH₃, N₃CH₂CH₂, NH₂, ANS, N₃, MeO, SH, Ph, F, PhNH, PhO,or RS (where Ph can be a phenyl group or phenyl ring, and F can be afluorine atom or group). The substituted groups can be in the S or Rconfiguration.

The non-incorporatable nucleotides can be alpha-phosphate modifiednucleotides, alpha-beta nucleotides, beta-phosphate modifiednucleotides, beta-gamma nucleotides, gamma-phosphate modifiednucleotides, caged nucleotides, or di-nucleotides.

Many examples of non-incorporatable nucleotides are known (Rienitz 1985Nucleic Acids Research 13:5685-5695), including commercially-availableones from Jena Bioscience (Jena, Germany).

Nanoparticles

Provided herein are mutant polymerases operably linked to at least onesuitable nanoparticle. The suitable nanoparticle can serve as a donorfluorophore in energy transfer reactions such as FRET. In oneembodiment, a semiconductor nanoparticle can be linked to a site nearthe nucleotide binding site on the mutant polymerase, to facilitateenergy transfer signals to/from the transiently-bound labelednucleotides.

In one aspect, the nanoparticle can be a core/shell nanoparticle whichtypically comprises a core surrounded by at least one shell. Forexample, the core/shell nanoparticle can be surrounded by an inner andouter shell. In another aspect, the nanoparticle is a core nanoparticlewhich has a core but no surrounding shell. The outmost shell istypically coated with tightly associated ligands that are not removed byordinary solvation.

The nanoparticle includes the core, shell(s), and ligand coatings.Methods for making core nanoparticles, core/shell nanoparticles, andligand coated nanoparticles are well known in the art.

In one aspect, the nanoparticle core and shell can be made from anysuitable metal and/or non-metal atoms for forming semiconductornanoparticles. The core and shell can be composed of differentsemiconductor materials.

The core can be composed of a semiconductor material (including ternaryand quaternary mixtures thereof), from: Groups II-VI of the periodictable, including ZnS, ZnSe, ZnTe, CdS, CdSe, CdTe, HgS, HgSe, HgTe,MgTe; Groups III-V, including GaN, GaP, GaAs, GaSb, InN, InP, InAs,InSb, AlAs, AlP, AlSb, AlS; and/or Group IV, including Ge, Si, Pb.

The shell can be composed of materials (including ternary and quaternarymixtures thereof) comprising: ZnO, ZnS, ZnSe, ZnTe, CdO, CdS, CdSe,CdTe, MgS, MgSe, GaAs, GaN, GaP, GaAs, GaSb, HgO, HgS, HgSe, HgTe, InAs,InN, InP, InSb, AlAs, AlN, AlP, or AlSb. The nanoparticle may be a dopedmetal oxide nanoparticle.

In one aspect, the dimensions of the nanoparticle can be, in theirlargest dimensions, about 50-100 nm, about 40-50 nm, about 30-40 nm,about 20-30 nm, about 15-20 nm, about 10-15 nm, about 5-10 nm, or about1-5 nm.

In one aspect, the nanoparticles can have different shapes, each ofwhich has distinctive properties including spatial distribution of thesurface charge; orientation dependence of polarization of the incidentlight wave; and spatial extent of the electric field. The shapes includespheres, rods, discs, triangles, nanorings, nanoshells, tetrapods,nanowires and so on.

In one aspect, the nanoparticle can be a semiconductor nanoparticlehaving size-dependent optical and electronic properties. For example,the nanoparticle can emit a fluorescent signal in response to excitationenergy. The spectral emission of the nanoparticle can be tunable to adesired energy by selecting the particle size, size distribution, and/orcomposition of the semiconductor nanoparticle. For example, depending onthe dimensions, the semiconductor nanoparticle can be a fluorescentnanoparticle that emits light in the UV-visible-IR spectrum. The shellmaterial can have a bandgap greater than the bandgap of the corematerial. Nanoparticles are available

In another aspect, the nanoparticle can be a fluorescent nanoparticle.The nanoparticles can be excited by an electromagnetic source such as alaser beam, or multi-photon excitation, or electrical excitation. Theexcitation wavelength can range between 190-800 nm including all valuesand ranges there between. The signal emitted by the nanoparticle can bebetween about 400-800 nm. In certain aspects, the nanoparticle can beexcited by an energy source having a wavelength of about 405 nm. Inother aspects, in response to excitation, the nanoparticles can emit afluorescent signal at about 605 nm.

As with common energy transfer donors, the efficiency of energy transfer(e.g., FRET) of a nanoparticle can depend sharply upon thedonor-acceptor distance R as 1/R⁶. The distance where FRET efficiency is50% is termed R₀, also known as the Förster distance. R₀ is unique foreach donor-acceptor combination and may be 5 to 10 nm. For nanoparticlesthat are size-tuned to emit in the visible light spectrum, the radiusfrom the nanoparticle's energy transferring core to its surfacetypically ranges from 2 to 5 nm. Given typical R₀ distances of 5-10 nm,this means that acceptor chromophores must be within a few nanometers ofthe nanoparticle surface for efficient FRET between donor-acceptorpairs.

Various types of core-shell nanoparticles are commercially-available.For example, InP/ZnS, CdS, CdSe, CdSe/ZnS, and D-dots™ are availablefrom NN-Labs. CdSe/ZnS Lumidots™ are available from Aldrich MaterialsScience in collaboration with Nanoco Technologies. Aggregatednanoparticles, such as TriLite™ nanoclusters containing about 8-12individual nanoparticles are available from Crystal Plex. Thesenanoclusters are 40-50 nm in size and are functionalized on the surfacewith carboxyl groups. Various QDOTS conjugated with streptavidin thatemit light in the range of about 525-800 nm, as well as severalbiotin-labeled QDOTS are available from Invitrogen. Also, Qdot™ ITK™nanoparticles that have an amphiphilic polymer coating andfunctionalized surface chemistry (carboxyl, amino-PEG, and organicsoluble groups) for custom conjugation to any desired molecule, areavailable from Invitrogen.

Other types of nanoparticles can be used. One example includesnanoparticles comprising a multi-shell layered core which is achieved bya sequential shell material deposition process, where one shell materialis added at a time, to provide a nanoparticle having a substantiallyuniform shell of desired thickness that is substantially free ofdefects. The nanoparticles can be prepared by sequential, controlledaddition of materials to build and/or applying layers of shell materialto the core. The methods can include at least one coordinating solvent.See e.g., U.S. provisional applications 61/108,425, Treadway, et al.,filed Oct. 24, 2008, and 61/144,613, Treadway, et al., filed Jan. 14,2009.

In one aspect, at least one coordinating solvent can be atrialkylphosphine, a trialkylphosphine oxide, phosphonic acid, or amixture of these. In another aspect, at least one coordinating solventcomprises trioctylphosphine (TOP), trioctylphosphine oxide (TOPO),tetradecylphosphonic acid (TDPA), or a mixture of these. In yet anotheraspect, the coordinating solvent comprises a primary or secondary amine,for example, decylamine, hexadecylamine, or dioctylamine.

In one aspect, the first inner shell precursor is Cd(OAc)₂ and thesecond inner shell precursor is bis(trimethylsilyl)sulfide (TMS₂S). Inother aspects, the first and second inner shell precursors are added asa solution in trioctylphosphine (TOP). In other aspects, the first outershell precursor is diethylzinc (Et₂Zn) and the second inner shellprecursor is dimethyl zinc (TMS₂S). Sometimes, the first and secondouter shell precursors are added as a solution in trioctylphosphine(TOP).

In one aspect, the nanoparticles can have ligands that coat the surface.The ligand coating can comprise any suitable compound(s) that providesurface functionality, including facilitating aqueous-dispersibility ofthe nanoparticles, or permitting binding and/or other interaction with abiomolecule. For example, the surface ligands can be: lipids;phospholipids; fatty acids; polynucleic acids; polyethylene glycol;primary antibodies; secondary antibodies; antibody fragments; protein ornucleic acid based aptamers; biotin; streptavidin; proteins; peptide;small organic molecules; organic or inorganic dyes; or precious or noblemetal clusters. Examples of ligands include: amphiphilic polymer (AMP);dihydrolipoic acid (DHLA); tetradecylphosphonic acid (TDPA);octylphosphonic acid (OPA); dipeptides (e.g., His-Leu and Gly-His);alkyl phosphonate; phosphine cross-linker (e.g.,tris(hydroxymethyl-phosphine; THP); L-carnosine; imidazole;4-aminobenzophenone; tris(hydroxymethyl)phosphine; and PEG (e.g.,PEG-1000 or amino-dPEG24-acid). See, e.g., U.S. Provisional Applications61/086,750; 61/102,709; 61/102,683; 61/102,666.

In one aspect, the nanoparticle comprises a core comprising CdSe. Inanother aspect, the nanoparticle shell can comprise YZ wherein Y is Cdor Zn, and Z is S, or Se. In one embodiment, at least one inner shelllayer comprises CdS, and the at least one outer shell layer comprisesZnS.

In one aspect, the nanoparticles exhibit modulated blinking properties,such as limited or no detectable blinking. The nanoparticles can have astochastic blinking profile in a timescale that is shifted to very rapidblinking or very slow or infrequent blinking relative to a nanoparticlepreviously described in the art. For example, the nanoparticles mayblink on and off on a timescale that is too rapid to be detected underthe methods employed to study this behavior.

In one aspect the nanoparticles are photostable. The nanoparticles canexhibit a reduced or no photobleaching with long exposure to moderate tohigh intensity excitation source while maintaining a consistent spectralemission pattern.

In one aspect, the nanoparticles have a consistently high quantum yield.For example, the nanoparticles can have a quantum yield greater thanabout 40%, or greater than about 50%, or greater than about 60%, orgreater than about 70%, or greater than about 80%.

In one embodiment, the nanoparticles can include a CdSe core. In anotheraspect the nanoparticles can include a CdS inner shell. In yet anotheraspect, the nanoparticles can include a ZnS outer shell.

In one embodiment, the spherical nanoparticles can be about 10 nm orsmaller. In another embodiment, the rod-shaped nanoparticles can beabout 12 nm or smaller, in their largest dimension. In one embodiment,the nanoparticles can include a ligand coating comprising: L-carnosine;dipeptides (e.g., His-Leu and/or Gly-His); 4-aminobenzophenone; citricacid; glycine; tris(hydroxymethyl)phosphine; and amino-dPEG24-acid.

It has been previously shown by others that nanoparticles producefluorescent signals in a variety of aqueous solutions, including purewater, various buffer solutions, and weakly acidic buffers. Using asingle-particle counting procedure, Zhang showed that acidic buffersdecrease the fluorescent intensity, but the burst count was not affected(Zhang 2008 Journal of the American Chemical Society 130:3750-3751).

The suitable nanoparticles include those described in U.S. Ser. No.61/086,750, having a 371(c) filing date of Aug. 6, 2008; 61/108,425having a 371(c) filing date of Oct. 24, 2008; 61/102,631, having a371(c) filing date of Oct. 3, 2008; 61/102,642, having a 371(c) filingdate of Oct. 3, 2008; 61/102,709, having a 371 (c) filing date of Oct.3, 2008; and 61/144,613, having a 371(c) filing date of Jan. 14, 2009.

Methods for Nucleotide Transient-Binding

Provided herein are methods for transiently binding a nucleotide to amutant polymerase. These methods are conducted under any reactioncondition which permits the mutant polymerase to selectively bind acomplementary nucleotide, but where incorporation of the complementarynucleotide is perturbed, impeded, or inhibited. Such reaction conditionsinclude utilizing: (1) any reaction conditions and reagents, such astemperature, pH, ionic strength, multivalent cations, and/or time; (2)non-incorporatable nucleotides; and/or (3) a non-extendiblepolymerization initiation site. Any combination of these reactionconditions can be practiced in any order in the transient-bindingmethods provided herein.

a) Temperature, pH, Ionic Strength

In one aspect, the methods include any reaction conditions and reagents,such as temperature, pH, and/or ionic strength. For example, thetransient-binding reactions can be conducted at a pH range whichinhibits polymerase-dependent nucleotide incorporation, such as a pHrange of about 4-12, or about 4-10, or about 4-8, or about 4-7.5, orabout 4-7, or about 4-6, or about 6-7.5. In another example, thereaction can be conducted at reduced temperatures (e.g., between about4-25° C.), or elevated temperatures (e.g., between about 25-80° C.). Inanother example, the reaction can be conducted with increased ionicstrength.

b) Time

Other reaction conditions include reducing the time that a nucleotide iscontacted with the mutant polymerase to a time that is statisticallyinsufficient to incorporate more than 1 or 2 successive nucleotides(Lapidus, U.S. Pat. No. 7,169,560). The reduced contact time can beachieved by introducing the nucleotide to the incorporation reaction inrapid flow or wash steps.

c) Metal Cations—Reducing Concentrations of Catalytic Metal Ions

Without wishing to be bound to any theory, a two-metal ion mechanism hasbeen postulated for the phosphoryl transfer reaction of DNA polymerases.The postulate suggests that the catalytic metal ion site (site A)coordinates with the alpha phosphate group of a nucleotide bound to thepolymerase, and the B site metal group coordinates with the leavingphosphate groups (Beese and Steitz 1991 EMBO Journal 10:25-33; Steitzand Steitz 1993 Proc. Natl. Acad. Sci. USA 90:6498-6502; Steitz 1998Nature 391:231-232). Catalytic metal ions can include magnesium,manganese, cobalt, strontium, or barium

Accordingly, the reaction conditions can include a reduction, omission,or chelation of any metal ion which permits nucleotide incorporation.The reduction, omission, or chelation of divalent cations, such asmagnesium, manganese, cobalt, strontium, or barium, may inhibitnucleotide incorporation. Chelation includes any procedure which rendersthe divalent cation unavailable for the nucleotide incorporationreaction, including using EDTA and/or EGTA.

The selection of the metal cation for which the concentration will bereduced, omitted or chelated in the reaction conditions, may depend uponthe mutant polymerase and nucleotides to be used in thetransient-binding reaction. It is known that certain polymerases usemagnesium for catalyzing phosphoryl transfer of an incoming triphosphatenucleotide, such as rat polymerase-beta (H Pelletier 1994 Science264:1981-1903), human polymerase-beta (M R Sawaya 1997 Biochemistry36:11205-11212), RB69 polymerase (M Wang 2009 Biochemistry 48:2075-2086;H R Lee 2009 48:2087-2098), Klenow (C M Joyce 2008 Biochemistry47:6103-6116), and HIV reverse transcriptase (N Kaushik 1996Biochemistry 35:11536-11546; H P Patel 1995 Biochemistry 34:5351-5363).Additionally, it is known that certain polymerases exhibit a preferencefor unlabeled nucleotides in the presence of magnesium.

It has also been shown that certain DNA polymerases (e.g., phi29) usemanganese for incorporating nucleotide polyphosphates having four ormore phosphate groups (Kumar, U.S. Pat. No. 7,393,640). Other DNApolymerases, including FY7 polymerase, may use manganese for catalysis(Fuller, U.S. Pat. No. 7,264,934; and Fuller, WO/2007/048033). Stillother polymerases may use magnesium or manganese (Fuller, U.S. publishedpatent application No. 2008/0287305), or magnesium and manganese.

Thus, the use of certain combinations of the mutant polymerases andnucleotides may guide the selection of the metal cation(s) thatpermit/support nucleotide incorporation, and it's concentration to bereduced, omitted, or chelated, in order to inhibit nucleotideincorporation. For example, the transient-binding methods can beconducted using manganese, a mutant RB69 polymerase, and dye-labelednucleotides (e.g., nucleotides having 3-7 phosphates linked at theterminal phosphate group to a fluorophore via an intervening linkermoiety).

In one embodiment, the magnesium can be any magnesium compound includingMgCl₂. In another embodiment, the manganese can be a manganese compoundincluding MnCl₂. In one embodiment, the amount of manganese or magnesiumcompounds which permits nucleotide incorporation can be about 0.01-10mM, or about 0.01-5 mM, or about 0.01-3 mM, or about 0.01-2 mM, or about0.01-1 mM. In another embodiment, the amount of manganese or magnesiumcompounds which permits nucleotide incorporation can be about 0.01-5 mM,or about 0.05-5 mM, or about 0.1-5 mM, or about 0.2-5 mM, or about 0.3-5mM, or about 0.4-5 mM, or about 0.5-5 mM, or about 1-5 mM, or about 2-5mM, or about 3-5 mM, or about 4-5 mM, or about 2-10 mM.

d) Cations which Inhibit Nucleotide Incorporation

In still another example, the reaction conditions can include at leastone type of multivalent cation which permits transient-binding of thenucleotide to the mutant polymerase but inhibits incorporation of thebound nucleotide. The transiently-bound nucleotide can be acomplementary or non-complementary nucleotide. The reaction conditionscan include a period IV cation including: calcium, scandium, titanium,vanadium, chromium, iron, cobalt, nickel, copper, zinc, gallium,germanium, arsenic, and selenium. The reaction conditions can includeother multivalent cations, including rhodium or strontium. The period IVcation compound can be ZnCl₂, CuCl₂, CoCl₂, FeSO₄, or NiCl₂. It has beenpreviously shown that substituting calcium for magnesium and/ormanganese permits nucleotide binding to wild-type or mutant polymerase(e.g., Klenow), but inhibits nucleotide incorporation (Gangurde 2002Biochemistry 41:14552-14559). The transient-binding reaction conditionscan include calcium at about 0.1-50 mM, or about 0.1-40 mM, or about0.1-30 mM, or about 0.1-20 mM, or about 0.1-10 mM, or about 0.1-5 mM.The reaction condition can include calcium at about 1-20 mM, or about2-20 mM, or about 3-20 mM, or about 4-20 mM, or about 5-20 mM, or about6-20 mM, or about 7-20 mM, or about 8-20 mM, or about 9-20 mM, or about10-20 mM. In another embodiment, the transient-binding reactionconditions can include any calcium compound, including CaCl₂ or anucleotide which is complexed or bound with calcium.

e) Cations which Permit Incorporation

The transient-binding reaction can be followed by a separatereaction/step which permits nucleotide incorporation, which can includethe presence of any cation which permits nucleotide incorporation (e.g.,manganese and/or magnesium). Accordingly, the methods provided hereininclude a nucleotide transient-binding step, and a nucleotideincorporation step. The methods can include a nucleotidetransient-binding step, a detection step, and a nucleotide incorporationstep. The reaction conditions can include a nucleotide transient-bindingstep, a detection step, a washing step, and a nucleotide incorporationstep.

For example, during the nucleotide transient-binding step, the reactionconditions can include a reduced concentration, omission, or chelation,of any metal cation which permit nucleotide incorporation (e.g.,magnesium, manganese, cobalt, strontium, or barium), and includes atleast one multivalent cation which permits transient-binding butinhibits nucleotide incorporation (e.g., any period IV cation, includingcalcium, scandium, titanium, vanadium, chromium, iron, cobalt, nickel,copper, zinc, gallium, germanium, arsenic, and selenium; and includesrhodium and strontium).

In another example, the nucleotide transient-binding step can includecalcium to permit nucleotide binding but inhibit incorporation, followedby a nucleotide incorporation step which includes a manganese compound(Clark, U.S. published patent application No. 2009/0176233), a magnesiumcompound, and/or a combination of manganese and magnesium.

In another example, a chemical quench assay can be conducted withcalcium during the pulse step and magnesium during the chase step (Lee2009 Biochemistry 48:2087-2098). In a related example, a FRET/quenchassay can be conducted in the presence of calcium or magnesium (Joyce2008 Biochemistry 47:6103-6116) for testing Klenow fragment (cysteine744 mutant) labeled with IAEDNAS donor dye bound to primer/templatelabeled with DABCYL acceptor/quench dye.

In another example, the transient-binding reaction includes aconcentration of any metal cation which permits nucleotideincorporation, and a concentration of any multivalent cation whichpermits transient-binding but inhibits nucleotide incorporation. In oneembodiment, the A and B metal binding sites on the mutant polymerase areboth occupied by the multivalent cation, or one of the sites is occupiedby the multivalent cation.

f) Cations which Promote Ternary Complex Formation and/or Stability

In yet another example, the reaction conditions can include at least onetype of exchange-inert cation which is complexed with a nucleotide, topermit transient-binding of the nucleotide to the mutant polymerase andinducing ternary complex formation (or stabilizing the ternary complex),but inhibiting incorporation of the bound nucleotide. Thetransiently-bound nucleotide can be a complementary or non-complementarynucleotide.

During nucleotide polymerization events, the mutant polymerase can be inan open conformation prior to binding a nucleotide. Upon binding thecomplementary nucleotide, the mutant polymerase can change to a closedconformation (also known as the ternary complex). The ternary complexcan include the mutant polymerase (in a closed conformation) which isbound to the template nucleic acid molecule which is base-paired withthe polymerization initiation site, and the nucleotide. The mutantpolymerase, in a closed conformation, can catalyze incorporation of thebound nucleotide. It is known that some cation-nucleotide complexes(e.g., chromium-nucleotides) promote the formation and/or stability ofthe ternary complex.

The transient binding reactions can include at least one type of cationwhich promotes the formation and/or the stability of the ternarycomplex. These reactions can be conducted in the presence or absence ofcations that are required for catalysis (e.g., Mg²⁺ and/or Mn²⁺). Forexample, Cr(III).nucleotide complexes have been previously used asMn(II).nucleotide analogs when conducting exchange-inert reactions withpolymerases (Zhong, et al., 1998 Journal am Chem Soc 120:235-236). Zhongcomplexed DNA polymerase β from rat brain with DNA template/primerduplexes containing the 2-aminopurine nucleotide analog opposite of theincoming nucleotide insertion site, and reacted the polymerase-DNAbinary complex with a Cr(III).nucleotide complex in the absence of acatalytic metal cation (e.g., Mg²⁺). The Cr(III).nucleotide complexinduced the polymerase to form a ternary conformation without catalysis,as indicated by the change of the 2-aminopurine fluorescence.

In one embodiment, the transient binding reactions can be conducted witha mutant polymerase bound to a nucleic acid template molecule which isbase-paired with a polymerization initiation site and aCr(III).nucleotide complex (e.g., a complementary nucleotide) withoutMg²⁺ or Mn²⁺. The Cr(III).nucleotide complex can induce the formation ofa ternary complex. The presence of the bound Cr(III).nucleotide complex,and/or the identity of the base in the Cr(III).nucleotide complex, canbe detected if the Cr(III).nucleotide complex is labeled with afluorescent reporter moiety. A catalytic cation can be added (e.g., Mg²⁺or Mn²⁺) to induce cleavage and incorporation of the chromium-complexednucleotide.

The Cr(III).nucleotide complex can be a chromium monodentate, bidentate,or tridentate complex. The Cr(III).nucleotide complex can be anα-monodentate, or β-γ-bidentate nucleotide. The Cr(III).nucleotidecomplex can be prepared using any well known methods, including mixingtogether nucleotides (e.g., dATP, dGTP, dCTP, dTTP, or dUTP) withchromium (e.g., CrCl₃) at an elevated temperature (e.g., approximately80° C. (see Dunaway-Mariano and Cleland 1980 Biochemistry 19:1496-1505;Dunaway-Mariano and Cleland 1980 Biochemistry 19:1506-1515). The variousdiastereomers of the β-γ-bidentate can be separated using reverse-phaseHPLC techniques (Gruys and Schuster 1982 Analytical Biochemistry125:66-73). Characterization of the various diastereomers can be doneusing phosphorus NMR or mass spectrometry.

Non-Extendible Polymerization Initiation Site

In some embodiments, the nucleotide transient-binding methods can beconducted using a non-extendible polymerization initiation site. Theextendible polymerization initiation site can include a terminal 3′-OHgroup which serves as a substrate for the mutant polymerase to form aphosphodiester bond between the terminal 3′-OH group and an incomingnucleotide. The extendible polymerization initiation site can be theterminal 3′-OH group on a primer molecule, or an internal 3′-OH group ina nick or gap within a nucleic acid molecule. The non-extendiblepolymerization initiation site can be a terminal group which does notserve as a substrate for polymerase-dependent nucleotide incorporation.For example, the non-extendible polymerization initiation site can be aterminal nucleotide which lacks a terminal 3′-OH group, or includes asugar-linked 2′ or 3′ blocking group, or can include a base-linkedmoiety which inhibits extension by a given polymerase (e.g., mutantpolymerase), or can include a sugar- or base-linked moiety which isbulky or is negatively charged.

In one embodiment, the nucleotide transient-binding methods can beperformed by binding the mutant polymerase to a template molecule whichis base-paired with a polymerization initiation site having anon-extendible end. The mutant polymerase binds and interrogates acandidate nucleotide, but the candidate nucleotide is not incorporated.A signal from the transiently-bound candidate nucleotide is detected,and its identity is deduced. The non-extendible end can include adideoxynucleotide or a terminator nucleotide.

Strand extension can be performed if the non-extendible end of thepolymerization initiation site is modified or removed to provide anextendible end (e.g., de-blocking). When a terminator nucleotide isincorporated at the extendible end, it can provide a new non-extendibleend. The mutant polymerase binds and interrogates another candidatelabeled nucleotide, and a signal is detected from the transiently-boundnucleotide. And the steps can be repeated.

In some embodiments, the reaction conditions can include reducing thetemperature and/or pH, omitting manganese and/or magnesium, and/oradding calcium, or any combination thereof.

Identifying a Nucleotide Bound to a Polymerase:

Optionally, the identity of a transiently-bound nucleotide can bedetermined according to the methods provided herein. In one aspect,methods for identifying a nucleotide bound to a mutant polymerase,comprises the steps of: (a) contacting at least one type of a labelednucleotide to an immobilized complex having a first polymerase (e.g.,mutant polymerase) bound to a template nucleic acid molecule which isbound to a polymerization initiation site, under suitable conditions totransiently-bind the at least one type of labeled nucleotide to theactive site of the polymerase in a nucleic acid template-dependentmanner and to inhibit nucleotide polymerization by the polymerase; (b)exciting the labeled nucleotide with an excitation source; (c) detectinga signal, or a change in a signal, from the transiently-bound labelednucleotide; and (d) identifying the nucleotide transiently-bound to thepolymerase.

In one embodiment, the methods for identifying a nucleotide bound to amutant polymerase, further comprises the steps of: (e1) removing thetransiently-bound nucleotide; and (f1) contacting the complex with atleast one type of nucleotide under suitable conditions for thepolymerase to polymerize the nucleotide. In this embodiment, the samepolymerase in step (a1) is contacted in step (f1).

In another embodiment, the methods for identifying a nucleotide bound toa polymerase, further comprises the steps of: (e2) removing the firstpolymerase and the transiently-bound nucleotide so that the templatenucleic acid molecule, nucleic acid primer molecule or self-primingtemplate nucleic acid molecule remains immobilized to the surface; (f2)binding the remaining template nucleic acid molecule with a secondpolymerase; and (g2) contacting the second polymerase with at least onetype of nucleotide under suitable conditions for the second polymeraseto polymerize the nucleotide. In this embodiment, the polymerase in step(a2) is different from the polymerase in step (f2).

Nucleotide Transient-Binding Reactions I:

In one embodiment, a method for identifying a nucleotide bound to apolymerase, comprises the steps of: (a1) contacting at least one type ofa labeled nucleotide to an immobilized complex having a polymerase(e.g., mutant polymerase) bound to a template nucleic acid moleculewhich is bound to a polymerization initiation site, under suitableconditions to transiently-bind the at least one type of labelednucleotide to the polymerase in a nucleic acid template-dependent mannerand to inhibit nucleotide polymerization by the polymerase; (b1)exciting the first labeled nucleotide with an excitation source; (c1)detecting a signal, or a change in a signal, from the transiently-boundlabeled nucleotide; and (d1) identifying the nucleotidetransiently-bound to the polymerase; (e1) removing the transiently-boundnucleotide; (f1) contacting the complex with at least one type ofnucleotide under suitable conditions for the polymerase to polymerizethe nucleotide; and (g1) repeating steps (a1)-(e1)

Nucleotide Transient-Binding Reactions II:

In one embodiment, a method for identifying a nucleotide bound to apolymerase, comprises the steps of: (a2) contacting at least one type ofa labeled nucleotide to an immobilized complex having a polymerase(e.g., mutant polymerase) bound to a template nucleic acid moleculewhich is base-paired to a nucleic acid primer molecule or theimmobilized complex having a polymerase bound to a self-priming templatenucleic acid molecule, under suitable conditions to transiently-bind theat least one type of labeled nucleotide to the polymerase in a nucleicacid template-dependent manner and to inhibit nucleotide polymerizationby the polymerase; (b2) exciting the first labeled nucleotide with anexcitation source; (c2) detecting a signal, or a change in a signal,from the transiently-bound labeled nucleotide; and (d2) identifying thenucleotide transiently-bound to the polymerase; (e2) removing thepolymerase and the transiently-bound nucleotide so that the templatenucleic acid molecule, primer nucleic acid molecule or self-primingtemplate nucleic acid molecule remains immobilized to the surface; (f2)binding the remaining template nucleic acid molecule with a secondpolymerase; (g2) contacting the second polymerase with at least one typeof nucleotide under suitable conditions for the second polymerase topolymerize the nucleotide; and (h2) repeating steps (a2)-(f2).

a) Suitable Conditions for Nucleotide Transient Binding:

In one embodiment, the suitable conditions to transiently bind thenucleotide to the polymerase in step (a1) or (a2) comprise: (i) reducingthe levels or omission of a metal cation that permits nucleotideincorporation and/or addition of a cation that inhibits nucleotideincorporation; (ii) using a polymerase (e.g., mutant polymerase) whichselectively binds the nucleotide in a template-dependent manner andexhibits reduced nucleotide incorporation activity; (iii) using at leastone type of labeled nucleotide which can be a labeled non-incorporatablenucleotide; and/or (iv) using a polymerization initiation site which canbe a non-extendible polymerization initiation site. Any combination ofthese suitable conditions can be practiced to identify the nucleotidebound to the polymerase.

b) Suitable Conditions for Nucleotide Incorporation:

In another embodiment, the suitable conditions for polymerizing thenucleotide in step (f1) or (g2) comprise: (i) including a metal cationthat permits nucleotide incorporation and/or reducing the levels oromission of a cation that inhibits nucleotide incorporation; (ii) usinga polymerase (e.g., mutant polymerase) which selectively binds thenucleotide in a template-dependent manner and polymerizes the boundnucleotide; (iii) using at least one type of incorporatable nucleotide;and/or (iv) using a polymerization initiation site having an extendiblepolymerization initiation site.

c) Polymerization Initiation Site:

In one embodiment, the polymerization initiation site can be the 3′terminal end of a nucleic acid primer molecule or of self-primingtemplate nucleic acid molecule. In another embodiment, thepolymerization initiation site can be base-paired to the templatenucleic acid molecule. In another embodiment, the polymerizationinitiation site can be an extendible terminal 3′OH group or anon-extendible terminal group. In another embodiment, the polymerizationinitiation site can be a terminal 3′OH group of the nucleic acid primermolecule or a terminal 3′OH group of a self-priming template nucleicacid molecule.

d) Template Molecules:

In one embodiment, the template nucleic acid molecule can be a DNAmolecule, RNA molecule, or DNA/RNA hybrid molecule.

e) Immobilized Template Molecules:

In one embodiment, the template nucleic acid molecule, the nucleic acidprimer molecule, or self-priming template nucleic acid molecule can beimmobilized to a surface.

f) Cation:

In one embodiment, the cation that inhibits nucleotide incorporation canbe calcium, scandium, titanium, vanadium, chromium, iron, cobalt,nickel, copper, zinc, gallium, germanium, arsenic, selenium, rhodium, orstrontium.

g) Polymerases:

In one embodiment, the first polymerase binds the labeled nucleotide ina nucleic acid template-dependent manner and exhibits reduced nucleotideincorporation activity. In another embodiment, the first polymerase canbe a DNA-dependent, mutant RB69 polymerase. In one embodiment, themutant polymerase, under certain reaction conditions, can bindnucleotides but exhibits reduced nucleotide incorporation activity. Inanother embodiment, a suitable mutant polymerase can be selected thatcan bind the labeled nucleotide. In another embodiment, a suitablemutant polymerase can be selected that can bind the template nucleicacid molecule which can be base-paired to the polymerization initiationsite. In another embodiment, the polymerization initiation site caninclude a terminal 3′OH extendible end or a terminal 3′ non-extendibleend. In another embodiment, a suitable mutant polymerase can be selectedthat can bind an incorporatable or a non-incorporatable nucleotide. Instill another embodiment, the mutant polymerase can be operably linkedto a reporter moiety (e.g., energy transfer donor moiety). In anotherembodiment, the mutant polymerase in step (a) can be an RB69 (exo−)(FIGS. 2-8; SEQ ID NO:2-8, respectively).

In another embodiment, the second polymerase can be the same type or adifferent type as the first polymerase.

h) Labeled Nucleotides:

In some embodiments, the labeled nucleotide can include 3-10 or morephosphate groups. In another embodiment, the labeled nucleotide can beadenosine, guanosine, cytidine, thymidine or uridine, or any other typeof labeled nucleotide. In another embodiment, the mutant polymerase canbe contacted with more than one type of labeled nucleotide (e.g., A, G,C, and/or T/U, or others). In another embodiment, each type of labelednucleotide can be operably linked to a different reporter moiety topermit nucleotide identity. In another embodiment, each type of labelednucleotide can be operably linked to one type of reporter moiety. Inanother embodiment, the labeled nucleotides are operably linked at theterminal phosphate group with a reporter moiety. In another embodiment,the labeled nucleotides are operably linked at the base moiety with areporter moiety. In another embodiment, the labeled nucleotide can be anon-incorporatable nucleotide.

In some embodiments, the non-incorporatable nucleotide can bind to themutant polymerase and template nucleic acid molecule which can bebase-paired to a polymerization initiation site, in a template-dependentmanner, but does not incorporate. In one embodiment, different types oflabeled nucleotides can be employed in the method for detecting thepresence of a transiently-bound nucleotide in order to determine thefrequency, duration, or intensity, of a transiently-bound nucleotide.For example, a comparison can be made between thefrequency/duration/intensity of transiently-bound complementary andnon-complementary nucleotides. Typically, for direct excitation of thereporter moiety, the length of the transient binding time of acomplementary nucleotide can be longer and/or more frequent compared tothat of a non-complementary nucleotide. Typically, for FRET-basedexcitation and detection of the reporter moieties, the transient bindingtime of a complementary nucleotide can be of longer duration compared tothat of a non-complementary nucleotide.

i) Non-Incorporatable Nucleotides:

In one embodiment, the labeled nucleotide in step (a) can be a labelednon-incorporatable nucleotide. In another embodiment, the labelednon-incorporatable nucleotide can be an adenosine, guanosine, cytidine,thymidine, or uridine nucleotide.

j) The Labels:

In one embodiment, the label (on the incorporatable ornon-incorporatable nucleotides) can be an energy transfer acceptorreporter moiety. In another embodiment, the label can be a fluorescentdye. In another embodiment, the adenosine, guanosine, cytidine,thymidine, or uridine nucleotides can be operably linked to differenttypes of labels. In another embodiment, the complex can be contactedwith at least two types of labeled nucleotides in step (a). In anotherembodiment, the at least two types of nucleotides can have differenttypes of labels.

k) Energy Transfer Moieties:

In one embodiment, the mutant polymerase can be operably linked to anenergy transfer donor reporter moiety. In another embodiment, the energytransfer donor reporter moiety can be an inorganic nanoparticle or afluorophore. In another embodiment, the mutant polymerase can beoperably linked to an energy transfer donor reporter moiety and thetransiently-bound nucleotide can be operably linked to an energytransfer acceptor reporter moiety. In another embodiment, thetransiently-bound labeled nucleotide can emit a FRET signal. In anotherembodiment, the signal from the transiently-bound labeled nucleotide canbe optically detectable.

l) FRET Signals

In one embodiment, the mutant polymerase can be operably linked to anenergy transfer donor (e.g., fluorescent dye or nanoparticle). Inanother embodiment, the labeled nucleotide can have an energy transferacceptor moiety (e.g., fluorescent dye). In yet another embodiment, theenergy transfer donor and acceptor can be a FRET pair. In anotherembodiment, the signal (or change in the signal) from the energytransfer donor or acceptor can be used to detect the presence of thetransiently-bound nucleotide. In still another embodiment, the signalemitted by the transiently-bound nucleotide can be a FRET signal.

m) Chain Terminating Nucleotides:

In one embodiment, the non-extendible terminal group can be an inhibitormoiety which inhibits incorporation of a subsequent in-comingnucleotide. In another embodiment, the inhibitor moiety can be removablevia an enzymatic, heat, chemical, or light cleavage reaction.

n) Excitation Source and Signals:

In one embodiment, the excitation source can be electromagneticradiation. The excitation source can be a laser. The signal, or thechange in the signal, can be optically detectable. In one embodiment,the mutant polymerase has an active site. The active site can beenzymatically-active. The labeled nucleotide can bind the active site,thereby bringing the mutant polymerase and labeled nucleotide in closeproximity with each other. The mutant polymerase may be labeled orunlabeled. In one embodiment, the signal or change in the signal can bea fluorescent signal resulting from direct excitation of the label whichcan be operably linked to the transiently-bound labeled nucleotide or tothe labeled mutant polymerase. In one embodiment, the energy transferdonor and/or acceptor moieties can fluoresce in response to directexcitation. These fluorescence responses can be a signal or change in asignal. In another embodiment, the energy transfer acceptor moiety canfluoresce in response to energy transferred from a proximal excitedenergy transfer donor moiety. These fluorescence responses can be asignal or change in a signal.

The proximal distance between the donor and acceptor moieties thataccommodates energy transfer can be dependent upon the particulardonor/acceptor pair. The proximal distance between the donor andacceptor moieties can be about 1-20 nm, or about 1-10 nm, or about 1-5nm, or about 5-10 nm. In another embodiment, the energy transfer signalgenerated by proximity of the donor moiety to the acceptor moiety canremain unchanged. In another embodiment, the energy transfer signalgenerated by proximity of the donor moiety to the acceptor moietyresults in changes in the energy transfer signal. In another embodiment,the changes in the signal or the energy transfer signal from the donoror acceptor moiety can include changes in the: intensity of the signal;duration of the signal; wavelength of the signal; amplitude of thesignal; polarization state of the signal; duration between the signals;and/or rate of the change in intensity, duration, wavelength oramplitude. In another embodiment, the change in the signal or the energytransfer signal can include a change in the ratio of the change of theenergy transfer donor signal relative to change of the energy transferacceptor signals. In another embodiment, the signal or the energytransfer signal from the donor can increase or decrease. In anotherembodiment, the signal or the energy transfer signal from the acceptorcan increase or decrease. In another embodiment, the signal or theenergy transfer signal associated with nucleotide transient-bindingincludes: a decrease in the donor signal when the donor is proximal tothe acceptor; an increase in the acceptor signal when the acceptor isproximal to the donor; an increase in the donor signal when the distancebetween the donor and acceptor increases; and/or a decrease in theacceptor signal when the distance between the donor and acceptorincreases.

o) Detection:

In one embodiment, the detecting the signal or change in the signal canbe performed using confocal laser scanning microscopy, Total InternalReflection (TIR), Total Internal Reflection Fluorescence (TIRF),near-field scanning microscopy, far-field confocal microscopy,wide-field epi-illumination, light scattering, dark field microscopy,photoconversion, wide field fluorescence, single and/or multi-photonexcitation, spectral wavelength discrimination, evanescent waveillumination, scanning two-photon, scanning wide field two-photon,Nipkow spinning disc, and/or multi-foci multi-photon.

p) Identifying the Transiently-Bound Nucleotide:

In practicing the nucleotide transient-binding methods, non-desirablefluorescent signals can come from sources including background and/ornoise. In one embodiment, the desirable signals can be distinguishedfrom the non-desirable fluorescent signals by measuring, analyzing andcharacterizing attributes of all fluorescent signals generated by thenucleotide transient-binding reaction. In one embodiment, attributes ofthe signal that can permit distinction from the non-desirablefluorescent signals can include: duration; wavelength; amplitude; photoncount; and/or the rate of change of the duration, wavelength, amplitude;and/or photon count. In one embodiment, the identifying the signal,includes measuring, analyzing and characterizing attributes of:duration; wavelength; amplitude; photon count and/or the rate of changeof the duration, wavelength, amplitude; and/or photon count. In oneembodiment, identifying the signal can be used to identify thetransiently-bound nucleotide.

q) Washing Steps

In yet another embodiment, washing steps can be included after any ofthe steps, for example, to remove the non-incorporated nucleotidesand/or the labeled nucleotides. In the wash step, magnesium, manganese,or calcium can be omitted or included.

EXAMPLES

All of the compositions and methods disclosed and claimed herein can bemade and executed without undue experimentation in light of the presentdisclosure. In some cases, the compositions and methods of thisinvention have been described in terms of embodiments, however theseembodiments are in no way intended to limit the scope of the claims, andit will be apparent to those of skill in the art that variations may beapplied to the compositions and/or methods and in the steps or in thesequence of steps of the methods described herein without departing fromthe concept, spirit and scope of the invention. More specifically, itwill be apparent that certain components which are both chemically andphysiologically related may be substituted for the components describedherein while the same or similar results would be achieved. All suchsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

Example 1 Preparing Mutant RB69 Polymerase (3PDX)

The nucleotide sequence encoding the 3PDX DNA polymerase (FIG. 2, SEQ IDNO:2) was cloned into pTTQ expression vector (Invitrogen, Carlsbad,Calif.). The vector was transformed into T7 express B1-21 cell line (NewEngland Biolabs). The expressing cell line was grown in 2YT media at 37°C. (Invitrogen #22712-020). When the optical density of the growingculture reached 0.6 OD, the cells were induce with 0.5M IPTG, thenshifted to 15° C. and allowed to continue to grow overnight. Cells wereconcentrated by differential centrifugation, stored at −80° C.

Cells were resuspended in 50 mM TRIS pH 7.5, 50 mM Glucose, 0.1 mM EDTA(pH 8.0), 0.05% Tween-20 and 1 mM DTT, and ruptured by high pressureusing a Microfluidizer, ML-110PS, (Microfluidics). While stirring onice, the salt concentration of the lysate was increased to a finalconcentration of 1M NaCl. Streptomycin sulfate was added to the lysateto a final concentration of 0.2% and the lysate was stirred for 20minutes. Cell debris was removed by differential centrifugation. PEI(polyethylenime) was added to the lysate to a final concentration of 2%,while stirring on ice. The precipitated DNA was removed by differentialcentrifugation. The lysate was precipitated with ammonium sulfate at afinal concentration of 65%. The resulting pellets were stored at −20° C.until further processing.

The expressed protein was purified using four columns. The components ofthe column buffers included: Buffer A: 25 mM HEPES (pH 7.5), 0.1 mMEDTA, 0.05% Tween-20 and 1 mM DTT. Buffer B: 25 mM HEPES (pH 7.5), 0.1mM EDTA, 0.05% Tween-20, 1 mM DTT and 1M NaCl. The pellets from theammonium sulfate precipitation were resuspended in Buffer A until theconductivity reached ˜11 mS/cm. The sample was filtered with a 1 μmfilter (Acrodisc 1 μM Glass fiber membrane, 37 mm; PALL) and thecollected protein was purified by the following four columns: (1) EMDsulfite cation exchange column, (Merck chemicals); (2) Poros HQ20,10×100 mm column (Life Technologies); (3) Poros PI20 (10×100 mm) column(Life Technologies); and (4) PorosHE50 (10×100 mm) column (LifeTechnologies). The sample was loaded on the first column, the proteinswere eluted with a 20 column volume gradient from 0 to 100% Buffer B, (0to 1 M NaCl). The fractions containing the expressed protein wereidentified by SDS PAGE gel (NuPAGE, 10% denaturing gel, using MESBuffer). These fractions were pooled and applied to the next column.This method was repeated for the next three columns. After the finalcolumn, the fractions containing the purified protein were identifiedand pooled for dialysis in 10 mM Tris pH 7.5, 0.1 mM EDTA, 100 mM NaCland 50% glycerol. The protein was dialyzed overnight (10 MWCO).

The purified protein was concentrated four fold. The proteinconcentration was determined by UV280. The enzyme was assayed forexogenous DNAse contamination and polymerase activity. Protein Puritywas determined by SDS PAGE gel electrophoresis.

Example 2 Analyzing Polymerase Kinetics Using Stopped-Flow Spectrometer

The kinetics of nucleotide incorporation by Phi29 and RB69 polymeraseswere compared using stopped-flow spectrometry.

1) Phi29 Polymerase: Stopped Flow Measurements of t_(pol)

Template A1 sequence: (SEQ ID NO: 12)AF546-5′-CGTTCCACGCCCGCTCCTTTGCAAC-3′ Template T1 sequence:(SEQ ID NO: 13) AF546-5′-CGAACCTCGCCCGCTCCTTTGCAAC-3′Template C1 sequence: (SEQ ID NO: 14)AF546-5′-CGTTAACCGCCCGCTCCTTTGCAAC-3′ Template G1 sequence:(SEQ ID NO: 15) AF546-5′-CGTTAAGCGCCCGCTCCTTTGCAAC-3′ Primer sequence:(SEQ ID NO: 16) 5′-GTTGCAAAGGAGCGGGCG-3′

The kinetics of nucleotide incorporation by recombinant phi 29 (exo−)(HP-1) and RB69 (exo−) (SEQ ID NO:4) (see section 4 below) DNApolymerases were measured in an Applied Photophysics SX20 stopped-flowspectrometer by monitoring changes in fluorescence from ALEXA FLUOR546-labeled primer/template duplex following the mixing of theenzyme-DNA complex with dye-labeled nucleotides in the reaction buffercontaining 50 mM Tris-HCl, pH 7.5, 50 mM NaCl, 4 mM DTT, 0.2% BSA, and 2mM MnCl₂ or 5 mM CaCl₂. The reactions included 330 nM recombinant DNApolymerase, 100 nM template/primer duplex, and 7 μM labeled nucleotides.

The averaged (5 traces) stopped-flow fluorescence traces (>1.5 ms) werefitted with a double exponential equation (1) to extrapolate the ratesof the nucleotide binding and product release,

Fluorescence=A ₁ *e ^(−k1*t) +A ₂ *e ^(−kpol*t) +C  (equation 1)

where A₁ and A₂ represent corresponding fluorescence amplitudes, C is anoffset constant, and k1 and kpol are the observed rate constants for thefast and slow phases of the fluorescence transition, respectively. Thedye-labeled nucleotides comprise terminal-phosphate-labeled nucleotideshaving an alkyl linker with a functional amine group attached to thedye. The stopped-flow techniques for measuring t_(pol) (1/k_(pol))followed the techniques described by M P Roettger (2008 Biochemistry47:9718-9727; M. Bakhtina 2009 Biochemistry 48:3197-320). Representativestopped-flow fluorescence traces for Phi29 (exo−) polymerase andterminal phosphate labeled dN4P nucleotides in the presence of manganeseare shown in FIG. 9A, or in the presence of calcium (FIG. 9B).

2) Phi 29 Polymerase: Stopped Flow Measurements of t⁻¹

Template A2 sequence: (SEQ ID NO: 17)AF546-5′-CAGTCCAGGA GTT GGT TGG ACG GCT GCG AGG C-3′Template T2 sequence: (SEQ ID NO: 18)AF546-5′-CAGTAATGGA GTT GGT TGG ACG GCT GCG AGG C-3′Template C2 sequence: (SEQ ID NO: 19)AF546-5′-CAGTAACGG AGT TGG TTG GAC GGC TGC GAG GC-3′Template G2 sequence: (SEQ ID NO: 20)AF546-5′-CAGTAAGGGA GTT GGT TGG ACG GCT GCG AGG C-3′Dideoxy-primer sequence: (SEQ ID NO: 21)5′-GCC TCG CAG CCG TCC AAC CAA CTC ddC-3′ Primer sequence:(SEQ ID NO: 22) 5′-GCC TCG CAG CCG TCC AAC CAA CTC C-3′

The rate of the nucleotide dissociation (k⁻¹) from the ternary complexof [enzyme.DNA.nucleotide] was measured in an Applied Photophysics SX20stopped-flow spectrometer by monitoring changes in fluorescence fromALEXAFLUOR546-labeled primer/template duplex following the mixing of the[enzyme.DNA.labeled nucleotide] ternary complex with: (A) 50 μM cognatenon-labeled deoxynucleoside triphosphate in a buffer containing 50 mMTris-HCl (pH 7.5), 50 mM NaCl, 4 mM DTT, 0.2% BSA, and 2 mM MnCl₂, or(B) 50 μM cognate non-labeled deoxynucleoside triphosphate in a buffercontaining 50 mM Tris-HCl (pH 7.5), 50 mM NaCl, 4 mM DTT, 0.2% BSA, and5 mM CaCl₂ (for Phi29 (exo−) (HP-1) polymerase) or 10 mM CaCl₂ (for RB69(exo−) polymerase).

The ternary complexes were prepared using: 330 nM polymerase(Phi29(exo−) or RB69), 100 nM template/primer duplex, and 7 μM terminalphosphate-labeled nucleotides.

The averaged stopped-flow fluorescence traces (>1.5 msec) were fittedwith a single exponential equation (2) to extrapolate the rate of thenucleotide dissociation (k⁻¹) from the [enzyme.DNA.nucleotide] ternarycomplex.

Fluorescence=A ₁ *e ^(−k-1*t) +C  (equation 2)

where A₁ represents the corresponding fluorescence amplitude, C is anoffset constant, and k_(—1) and the observed rate constants for thefluorescence transition. The stopped-flow techniques for measuring t⁻¹(1/k⁻¹) followed the techniques described by M. Bakhtina (2009Biochemistry 48:3197-3208). Representative stopped-flow fluorescencetraces for Phi29 polymerase and terminal phosphate labeled dN4Pnucleotides in the presence of manganese are shown in FIG. 9C or in thepresence of calcium (FIG. 9D).

3) Phi29 Polymerase: Stopped-Flow Measurements for CharacterizingNucleotide Transient-Binding:

Stopped-flow procedures were conducted to characterize the t_(pol)(using 7 μM terminal phosphate labeled dG4P-AF647 and calcium) and t⁻¹rates (chased with 0.1 mM EDTA and 150 mM NaCl) of various polymerases.Representative stopped-flow fluorescence traces for Phi29 are shown inFIG. 9E. The results show that transient-binding of the nucleotide byPhi29 polymerase, in the presence of calcium, is reversible by chelatingthe calcium with the addition of EDTA.

Stopped flow procedures were conducted to characterize the selectivebinding of the correct nucleotides by various polymerases fortransiently-binding in the presence of calcium. The primer/templateduplexes were ALEXA FLUOR 546-labeled at the 5′ end. A representativestopped-flow fluorescent trace for Phi29 (5 mM CaCl₂, FIG. 9F), reactedwith one of four terminal phosphate labeled nucleotides (at 7 μM,labeled with AF647) is shown. The correct nucleotide is dG4P. Theresults show that Phi29 polymerase is selective in transient-binding thecorrect nucleotide, in the presence of calcium.

The apparent nucleotide dissociation constant (K_(d) ^(app)) for thePhi29 DNA polymerase in the presence of 2 mM calcium was measured in thestopped-flow procedure. A representative stopped-flow fluorescent tracefor Phi29 reacted with zero to 20 μM of the correct terminal phosphatelabeled nucleotide (dG4P-AF647) is shown in FIG. 9G.

The averaged individual stopped-flow fluorescent trace (>1.5 msec) shownin FIG. 9G were fitted to a hyperbola equation 3 (H Zhang 2007 NucleicAcids Research, pp. 1-11, doi:10.1093/nar/gkm587) to extrapolate theapparent nucleotide dissociation constant (K_(d) ^(app)) for the Phi29DNA polymerase.

$\begin{matrix}{{Fluorescence} = \frac{F_{\max} \cdot \lbrack{nucleotide}\rbrack}{K_{d}^{app} + \lbrack{nucleotide}\rbrack}} & \left( {{equation}\mspace{14mu} 3} \right)\end{matrix}$

where F_(max) represents the maximum fluorescence change, [nucleotide]is the concentration of nucleotide in the reaction, and K_(d) ^(app) isthe apparent nucleotide dissociation constant.

The fitted data is shown in FIG. 9H. For Phi29 (exo−) polymerase, in thepresence of 2 mM CaCl₂, the K^(app) _(d) is about 0.38±0.03 μM.

4) RB69 Polymerase: Stopped Flow Measurements of t_(pol)

Stopped-flow measurements of t_(pol) were conducted as described in thesection above, using the A1, C1, T1 or G1 oligo-template sequences, andthe same reaction conditions (see Example 3, section 1 above).Representative t_(pol) stopped-flow fluorescence traces for RB69polymerase in the presence of 2 mM manganese (FIG. 10A) or 10 mM calcium(FIG. 10B) are shown. The results show that RB69 polymerase binds thecorrect nucleotide but does not catalyze nucleotide incorporation in thepresence of manganese (FIG. 10A).

5) RB69 Polymerase: Stopped Flow Measurements of t⁻¹

Stopped-flow measurements of t⁻¹ were conducted as described in thesection above, using the A2, C2, T2, or G2 oligo-template sequences, andthe same reaction conditions (see Example 3, section 2 above).Representative t⁻¹ stopped-flow fluorescence traces for RB69 polymerasein the presence of manganese (2 mM, FIG. 10C) or calcium (10 mM, FIG.10D) are shown.

6) RB69 Polymerase: Stopped-Flow Measurements for CharacterizingNucleotide Transient-Binding:

Stopped flow procedures were conducted to characterize the selectivebinding of the correct nucleotides by RB69 polymerase fortransiently-binding in the presence of calcium, as described above usingthe primer/template duplexes which were ALEXA FLUOR 546-labeled at the5′ end (see Example 3, section 3 above). A representative stopped-flowfluorescent trace for RB69 polymerase (10 mM CaCl₂, FIG. 10E), reactedwith one of four terminal phosphate labeled nucleotides (at 7 μM,labeled with AF647) is shown. The correct nucleotide is dG4P. Theresults show that RB69 polymerase is selective in transient-binding thecorrect nucleotide, in the presence of calcium.

Example 3 Transient Binding Methods Using a Mutant RB69 Polymerase

In this example, the nucleotide transient-binding reaction andnucleotide incorporation reactions were conducted with a mutant RB69polymerase (3PDX), using a donor-labeled hairpin template, andacceptor-labeled nucleotides. The nucleotide incorporation reactionswere conducted with unlabeled 3′-azidomethyl terminator nucleotides.

Hairpin template sequence: (SEQ ID NO: 11)5′-Cy3-CAGTCTCGGGATCTTGTGCCATT(biotin-dT)TTTGGCA CAAGATCCC-3′.

Wash Buffer: 50 mM Tris pH 7.5 (Invitrogen PN 15567-027), 50 mM NaCl,0.2% Bovine Serum Albumin (Sigma PN A8577).

Reset Buffer: 50 mM Tris pH 7.5, 50 mM EDTA, 1 M NaCl, 0.2% BSA.

Imaging Solution: 50 mM Tris pH 7.5, 50 mM NaCl, 0.2% BSA, 1 uM 3PDXpolymerase, 200 nM base-labeled nucleotides (AF647-aha-dUTP (Invitrogen,A32763), Cy5-propargylamine-dGTP (Jena Biosciences, NU-1615-CY5),AF5-propargylamine-dATP, or AF647-aha-dCTP (Invitrogen, A32771)), 0.4%glucose, 2 mM Trolox (Fluka PN 56510), 2-5 mM CaCl₂, 0.2 mg/mL glucoseoxidase (Sigma PN G7141), 4 unit/μL Katalase (Fluka PN 02071).

1) Template Immobilization:

Flow chambers were assembled using PEG-biotin coated glass coverslips(Microsurfaces Inc., BIO 01). The flow chambers were washed with 1 mL ofWash Buffer. The flow chambers were injected with 5 nM streptavidin (inWash Buffer) and incubated for 20 minutes. The flow chambers were rinsedwith 1 mL Wash Buffer. The flow chambers were injected with 50 pMbiotin-tagged DNA-Cy3 into the flow and incubated for 10 minutes. Theflow chambers were washed with 1 mL Wash Buffer.

2) Nucleotide Transient-Binding Reaction:

The flow chambers were washed with 1 mL Wash Buffer. The flow chamberswere injected with Imaging Solution containing one of the fourdye-labeled nucleotides (e.g., dUTP), incubated for 3 minutes at roomtemperature, and imaged. Imaging was conducted by exciting at 532 nm at50 W/cm². The acceptor signals were detected at 670 nm with a bandwidthof 30 nm. The 3 minute incubating step, and the imaging step, wasrepeated twice for a total of three incubation and three imaging stepsfor each type of labeled nucleotide. The flow chambers were not washedprior to injecting and imaging the other types labeled nucleotides. Forthe other three types of nucleotides (e.g., dATP, dGTP, dCTP), the flowchambers were injected with Imaging Solution containing dye-labelednucleotides, and the incubating and imaging steps were repeated.

3) Nucleotide Incorporation:

The flow chambers were washed with 1 mL Reset Buffer. The flow chamberswere washed with 1 mL Wash Buffer. The flow chambers were injected with100 ul THERMINATOR Buffer (NEB, PN B9004S). The flow chambers wereinjected with 60 ul reversible terminator reaction mix containing fourtypes of nucleotides having the 3′ position of the sugar moiety blockedwith an azidomethyl group; the reversible terminator reaction mixcontained: 5 uM 3′-AM-dATP, 5 uM 3′-AM-dGTP, 2.5 uM 3′-AM-dCTP, 2.5 uM3′-AM-dTTP, lx THERMINATOR Buffer (NEB, PN B9004S), and 3 uM 3PDXpolymerase. The flow chambers were incubated at room temperature for 45minutes. The flow chambers were washed with 1 mL Reset Buffer. The flowchambers were washed with 1 mL Wash Buffer. The flow chambers wereinjected with 85 ul of 100 mM TCEP (Tris(2-carboxyethyl)phosphine) in0.1 M Tris-HCL at pH 7.5, and allowed to incubate at room temperaturefor 20 minutes. The flow chambers were washed with 1 mL Reset Buffer.The nucleotide transient-binding and nucleotide incorporation reactionswere repeated four times.

Mutant polymerases having the amino acid sequence of SEQ ID NO: 2, SEQID NO: 3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 and SEQ IDNO:8 were separately tested for nucleotide binding and nucleotideincorporation activity in assays conducted according to theabove-described procedures. The results indicated that some of thesemutant polymerases (e.g., 3PDX) exhibited increased duration ofnucleotide binding and increased nucleotide incorporation activityrelative to wild type RB69 polymerase (data not shown).

What is claimed:
 1. An isolated nucleic acid comprising a nucleotidesequence encoding a mutant DNA polymerase having the amino acid sequenceof SEQ ID NO:2.
 2. A vector comprising the nucleic acid sequence ofclaim
 1. 3. The vector of claim 2, further comprises a promoter sequencewhich is operably joined to the nucleic acid sequence encoding themutant DNA polymerase.
 4. The vector of claim 3, wherein the promoter isconstitutive or inducible.
 5. A host cell carrying the vector of claim2.
 6. A host cell carrying the vector of claim
 3. 7. A method forproducing a mutant DNA polymerase polypeptide, comprising culturing thehost cell of claim 6 under conditions suitable for the host cell toproduce the mutant DNA polymerase polypeptide.
 8. The mutant DNApolymerase produced by the method of claim 7.